Validation of commercially available sphingosine kinase 2 antibodies for use in immunoblotting, immunoprecipitation and immunofluorescence

Antibody details

The following SK2 antibodies were assessed: rabbit polyclonal anti-SK2 (ECM Biosciences; anti-Sphingosine Kinase 2 (N-terminal region); #SP4621, lot #1) and rabbit polyclonal anti-SK2 (Proteintech Group, Inc; anti-SPHK2; #17096-1-AP, lot #00010361). The ECM Biosciences SK2 antibody was raised against a synthetic peptide coupled to keyhole limpet hemocyanin (KLH), corresponding to amino acids 36–52 of human SK2a, and was affinity purified with the SK2 peptide (without KLH). It is reported by the manufacturer to have cross-reactivity with rat and mouse SK2 [human, mouse and rat SK2 share 100% sequence identity in this region (determined using the align tool and protein sequences from www.uniprot.org)], and has been assessed by the manufacturer for use in IB and enzyme-linked immunosorbent assay (ELISA). The Proteintech SK2 antibody was raised against truncated recombinant GST-tagged human SK2a (amino acid residues 266–618 generated in Escherichia coli using the PGEX-4T plasmid). The SK2 target antibodies were then affinity purified using 6xHis-tagged antigen protein (to remove GST-specific antibodies) and then again with the immunising GST-tagged antigen protein. It is reported to have cross-reactivity with rat and mouse SK2 [80.2% sequence identity between human and mouse SK2, and 80.2% sequence identity between human and rat SK2 in this region (determined using the align tool and protein sequences from www.uniprot.org)], and according to the manufacturer can be employed for IB, ELISA, IP and immunohistochemistry. Mouse anti-α-tubulin (DM1A; Abcam; #ab7291) is a mouse monoclonal antibody, which was used as a loading control for IB analyses, at a dilution of 1:5,000. All antibody details, including information for secondary antibodies used, are provided in Table 1.

Cell culture

Human embryonic kidney (HEK) 293 cells (CellBank Australia; #85120602) and HeLa human cervical cancer cells (ATCC; #CCL-2) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Thermo Fisher Scientific Inc.), containing 10% heat-inactivated fetal bovine serum (FBS; Bovagen), 1 mM HEPES, penicillin (1.2 mg/ml) and streptomycin (1.6 mg/ml). Cells were grown at 37°C with 5% CO₂ in a humidified incubator. Primary mouse embryonic fibroblasts (MEFs) were generated from both wildtype (WT) C57/Bl6 and Sphk2^-/- C57/Bl6¹⁷ mouse embryos at 14.5 days post coitum. The fibroblasts were isolated and cultured as described above, except they were maintained at 37°C in a humidified atmosphere with 10% CO₂.

RNAi knockdown of SK2

siRNA-mediated knockdown of SK2 was performed using human SPHK2 siGENOME SMARTpool siRNA (Dharmacon), which targets the following sequences: CCACUGCCCUCACCUGUCU, GCUCCUCCAUGGCGAGUUU, GAGACGGGCUGCUCCAUGA, CAAGGCAGCUCUACACUCA. Cells were seeded and grown to a cell density of approximately 50%, and were then transfected with 30 nM (final concentration) of either human SK2 siRNA or siGENOME non-targeting siRNA control pool (Dharmacon), using Lipofectamine RNAiMAX (Life Technologies), as per the manufacturer’s protocol. Cells were incubated with the siRNA complexes at 37°C for 48 h.

Immunoblotting

Specific details for all reagents used can be found in Table 2. Cells were pelleted by centrifugation (400 × g, 5 min, 4°C) and washed in cold phosphate buffered saline (PBS). Cell pellets were resuspended in extraction buffer [EB; 50 mM Tris/HCl buffer (pH 7.4) containing 150 mM NaCl, 10% glycerol, 1 mM EDTA, 0.05% Triton X-100, 2 mM Na₃VO₄, 10 mM NaF, 10 mM β-glycerophosphate, 1 mM dithiothreitol (DTT) and protease inhibitor cocktail (Roche)], and lysed by bath sonication (4× 30 sec on/off). Lysates were clarified (17,000 × g, 15 min, 4°C) and equal amounts of protein [as determined by a Bradford protein assay (Bio-Rad Laboratories)] were mixed with 5× Laemmli sample buffer, boiled at 100°C for 5 min, and separated by SDS-PAGE on a Criterion™ XT Bis-Tris 4–12% gradient gel (Bio-Rad Laboratories). Proteins were then transferred to nitrocellulose membrane (Pall Life Sciences) at 400 mA for 1 h. Membranes were blocked with 5% skim milk in PBS containing 0.1% Triton X-100 (PBS-T) for 1 h at room temperature, with gentle rocking. Membranes were probed with rabbit anti-SK2 antibodies diluted in Signal Boost primary antibody diluent at 1:1,000 (ECM Biosciences: 1 µg/ml; Proteintech: 687 ng/ml) overnight at 4°C with gentle rocking. Alternatively, membranes were probed with mouse anti-α-tubulin antibody diluted in 5% skim milk in PBS-T at 1:5,000 (200 ng/ml) for 1 h at room temperature, with gentle rocking. Following primary antibody incubation, membranes were washed 3 × 5 min in 5% skim milk in PBS-T at room temperature with gentle agitation. Membranes were probed with goat anti-rabbit horseradish peroxidase (HRP) secondary antibody diluted in Signal Boost secondary antibody diluent at 1:10,000 (40 ng/ml), or goat anti-mouse HRP secondary antibody diluted in 5% skim milk in PBS-T at 1:10,000 (40 ng/ml), for 1 h at room temperature, with gentle rocking. Membranes were washed 3 × 5 min in 5% skim milk in PBS-T at room temperature with gentle agitation, and proteins were visualized using enhanced chemiluminescence (ECL) on a LAS-4000 luminescence image analyser (Fujifilm). Exposure times are indicated in the figure legends for each blot.

Immunoprecipitation

HEK293 cell lysates were prepared as for immunoblotting, with the exclusion of DTT in the extraction buffer (EB–DTT). Protein concentration was determined from clarified lysates, and 800 µg total protein was transferred to fresh tubes and made up to 300 µl in EB–DTT. In total, 20 µl of diluted lysate was removed and mixed with 5× Laemmli sample buffer for immunoblot analysis. Immunoprecipitation was performed using the µMacs magnetic system (Miltenyi Biotec; see Table 3 for reagent details). Rabbit anti-SK2 antibodies (4 µg; ECM Biosciences 1:75 or Proteintech 1:52) or rabbit IgG isotype control antibody (4 µg), as well as 50 µl each of Protein A and G µBeads were added to the lysate, mixed gently and incubated on ice for 30 min. µMacs columns were placed onto a magnetic stand, equilibrated with 200 µl EB–DTT, and lysate/antibody/bead complexes were run through the columns. Columns were washed four times with 200 µl EB–DTT, and once with 100 µl low salt wash buffer, before the addition of 20 µl hot 1× Laemmli sample buffer for 5 min. Immunoprecipitates were then eluted with 50 µl hot 1× Laemmli sample buffer and collected in fresh tubes. Samples were boiled and 25 µl was analysed by SDS-PAGE and immunoblotting as described above.

Immunofluorescence staining

Cell lines were seeded onto coverslips coated with poly-L-lysine (Sigma-Aldrich) in a 12-well plate (HEK293: 1.5 ×10⁵ cells/well; HeLa: 7.5 ×10⁴ cells/well; MEF: 3 ×10⁴ cells/well). Cells were allowed to bed down overnight at 37°C in DMEM with 10% FBS. The following day (for HEK293 and HeLa cells), cells were treated with siRNA as described above, and incubated for 48 h. The following immunofluorescence staining protocol was then performed, with all steps carried out at room temperature (see Table 4 for reagent details). Cells were washed once in PBS, fixed in 4% paraformaldehyde for 10 min, washed three times in PBS-T, and permeabilized for 10 min in PBS-T. Cells were then blocked in 3% bovine serum albumin (BSA) in PBS-T for 30 min, and incubated for 1 h with anti-SK2 antibodies (4 µg/ml; ECM Biosciences 1:250 and Proteintech 1:172) diluted in 3% BSA/PBS-T. Cells were washed five times in PBS-T, and then incubated with goat anti-rabbit AlexaFluor 488 secondary antibody (1:500) for 1 h. After washing five times in PBS-T, cell nuclei were stained with DAPI (0.2 µg/ml) for 5 min. Cells were then washed twice in PBS, coverslips were partially dried and mounted onto slides using fluorescence mounting medium (Dako), and then left to set overnight. Fluorescence microscopy and imaging were performed using a Carl Zeiss LSM 700 confocal microscope, with Zen 2011 (Black Edition) version 8.1.5.484 software. All microscope settings, including gains, were kept constant for each cell line, allowing direct comparison between antibodies.

Controls

Various controls were used in these studies. Immunoprecipitations were performed with IgG isotype control antibody to control for any non-specific binding of proteins to the antibodies. Primary antibodies were also omitted from the immunofluorescence protocol to control for background fluorescence of the secondary antibody alone. siRNA-mediated SK2 knockdown as well as Sphk2^-/- MEFs were utilized to verify the specificity of the SK2 antibodies to their target.

Proteintech SK2 antibody demonstrates target specificity and sensitivity by immunoblot

Both SK2 antibodies examined in the present study are reported by their respective manufacturers to be able to detect endogenous SK2 by IB. To determine the selectivity of the anti-SK2 antibodies, we performed IB analyses using two human cell lines (HEK293 and HeLa) that had been treated with either scrambled control or SK2-directed siRNA, as well as WT and Sphk2^-/- MEFs. The Proteintech anti-SK2 antibody detected a single prominent band at the correct molecular weight for SK2 (~65 kDa), which was decreased or absent in the knockdown and knockout lines (Figure 1A; Dataset 1¹⁸). Some faint non-specific bands were also detected in both the WT and Sphk2^-/- MEF lysates by this antibody, which were not observed in the human cell lines. The ECM Biosciences anti-SK2 antibody did not appear to be very sensitive towards SK2, as no band was detected at the expected size in the HeLa lysates, and only very faint bands were present in the HEK293 and MEF lysates that were reduced or absent in the knockdown or knockout lines (Figure 1B). Furthermore, numerous prominent non-specific bands were present in all lysates, particularly in the MEF lines, indicating a lack of selectivity of this antibody towards SK2. Therefore, the Proteintech anti-SK2 antibody appears to be superior for use in IB, demonstrating both selectivity and sensitivity in the detection of endogenous SK2, particularly in the human cell lines tested.

Figure 1. Immunoblot analyses of endogenous SK2 in multiple cell lines using two commercially available rabbit polyclonal anti-SK2 antibodies.

Immunoblot analyses of lysates from HEK293 and HeLa cells treated with scrambled control siRNA (si-Neg) or SK2 siRNA (si-SK2), and lysates from wildtype (WT) or Sphk2^-/- MEFs. An equal amount (40 µg) of total protein from each sample was run in duplicate. After transferring to nitrocellulose and blocking, the membrane was separated and duplicate samples were probed with either (A) Proteintech rabbit anti-SK2 antibody or (B) ECM Biosciences rabbit anti-SK2 antibody. SK2 membranes were imaged using a 4 min exposure. The expected band size for SK2 is ∼65 kDa. Membranes were re-probed with mouse anti-α-tubulin antibody as a loading control (2 min exposure), which was detected at 55 kDa as expected. Consistent results were observed from 2-3 (HEK293 and MEF) or 3-4 (HeLa) independent experiments for each antibody.

ECM Biosciences SK2 antibody is able to specifically immunoprecipitate SK2

We also examined whether either of the commercial anti-SK2 antibodies could immunoprecipitate SK2 from cell lysates. The Proteintech anti-SK2 antibody is suggested by the manufacturer to be useful for IP, whereas to our knowledge the ECM Biosciences anti-SK2 antibody has not been previously tested for use in this application. Initially, using lysates from HEK293 cells, we found that the Proteintech anti-SK2 antibody was sometimes able to IP a band at the correct size for SK2 (Figure 2A); however this was not consistent with each experimental repeat and other proteins were also immunoprecipitated to a varying extent by this antibody that were not present in the IgG isotype control (Dataset 2¹⁹).

Figure 2. Comparison of two commercially available rabbit polyclonal anti-SK2 antibodies for immunoprecipitation of SK2 from HEK293 cell lysates.

SK2 was immunoprecipitated from HEK293 cell lysate using either (A) Proteintech rabbit anti-SK2 antibody or (B) ECM Biosciences rabbit anti-SK2 antibody. Normal rabbit IgG antibody was used as an isotype control. Immunoprecipitates (and 40 µg lysate input) were subjected to immunoblot analyses and probed with (A) Proteintech rabbit anti-SK2 antibody or (B) ECM Biosciences rabbit anti-SK2 antibody. Membranes were imaged using a 4 min exposure. Images are representative of three independent experiments for each antibody. (C) SK2 was immunoprecipitated from HEK293 cell lysates (of equal protein) that had been treated with scrambled control siRNA (si-Neg) or SK2 siRNA (si-SK2), using ECM Biosciences rabbit anti-SK2 antibody. Immunoprecipitates were subjected to immunoblot analyses and probed with ECM Biosciences rabbit anti-SK2 antibody. Membrane was imaged using a 4 min exposure. Image is representative of three independent experiments. IgG h/c = IgG heavy chain.

Conversely, the ECM Biosciences anti-SK2 antibody was able to consistently and cleanly IP a protein of the same size as SK2 from cell lysates, with almost no non-specific bands observed (Figure 2B; Dataset 2¹⁹). The protein immunoprecipitated by the ECM Biosciences antibody was considerably enriched from the cell lysate and was strongly detectable by this antibody, which was unable to detect SK2 in the lysate input sample, consistent with Figure 1B. To determine if this band was in fact SK2, the ECM Biosciences anti-SK2 antibody was then used to immunoprecipitate SK2 from HEK293 lysates treated with either scrambled control or SK2-directed siRNA. SK2 knockdown consistently resulted in reduced intensity of the band enriched by this antibody (Figure 2C; Dataset 2¹⁹), confirming that the ECM Biosciences anti-SK2 antibody can selectively IP endogenous SK2.

ECM Biosciences SK2 antibody can specifically detect SK2 by immunofluorescence staining

Finally, we examined whether these commercially available SK2 antibodies could selectively detect SK2 by IF. Neither antibody has been reported to be tested for use in IF by their respective manufacturers; however, the Proteintech SK2 antibody is recommended for immunohistochemistry. Using IF staining methods routinely performed in our laboratory, we compared the two anti-SK2 antibodies using HeLa, HEK293 and MEF cell lines. The Proteintech anti-SK2 antibody produced minimal staining in all cell lines tested (Figure 3A–C), and consequently there was no observable differences between the control cells and those with SK2 knockdown (in the human cell lines) or SK2 knockout (in the Sphk2^-/- MEF line).

Figure 3. Immunofluorescence staining analyses of endogenous SK2 in multiple cell lines using two commercially available rabbit polyclonal anti-SK2 antibodies.

(A) HeLa or (B) HEK293 cells were treated with scrambled control siRNA (si-Neg) or SK2 siRNA (si-SK2), and endogenous SK2 (green) was visualised by immunofluorescence staining and confocal microscopy, using Proteintech rabbit anti-SK2 antibody or ECM Biosciences rabbit anti-SK2 antibody. (C) Wildtype (WT) or Sphk2^-/- MEFs were seeded, and endogenous SK2 (green) was visualised by immunofluorescence staining and confocal microscopy, using Proteintech rabbit anti-SK2 antibody or ECM Biosciences rabbit anti-SK2 antibody. Nuclei were stained with DAPI (blue). For each cell line, background staining was examined by staining cells (si-Neg or WT cells) with secondary antibody and DAPI only, and collecting images using both 488nm and 405nm lasers (SK2 + DAPI). Images were taken at 40× magnification; scale bars = 10 µm. Images shown are representative of more than 100 cells from each experiment, and these results were consistent over three independent experiments for each cell line.

The ECM Biosciences anti-SK2 antibody did result in consistently observable staining in HeLa and HEK293 cells, which was substantially reduced upon knockdown of SK2 (Figure 3A and B; Dataset 3²⁰). Hence, in these cells the ECM Biosciences antibody was able to selectively detect SK2 by IF. Interestingly, in HeLa cells SK2 detected by the ECM Biosciences antibody was predominantly nuclear with some peri-nuclear/cytoplasmic localization, whereas in HEK293 cells SK2 was cytoplasmic and nuclear-exclusion, which is consistent with previous reports for these cell lines⁹. However, the ECM Biosciences anti-SK2 antibody produced very strong peri-nuclear staining/puncta in both the WT and Sphk2^-/- MEF lines (Figure 3C), suggesting that this staining was not specific for SK2 and represents non-specific binding to other proteins in this cell type. Increased non-specific binding of both SK2 antibodies to other proteins in the MEF lines was also observed when used for IB, so this cell type may not be suitable for use with these antibodies. It will remain to be determined if the same level of non-specificity is also observed in other mouse cell lines and tissues.