Multiple steps in membrane trafficking are coordinated by Rab proteins, a family of small guanosine triphosphatases (GTPase). ¹ Rab GTPases undergo a dynamic cycle, alternating between an active GTP-bound state, catalyzed by guanine exchange factors (GEF), and an inactive GDP-bound state, achieved through GTP hydrolysis, stimulated by a GTPase-activating protein (GAP). ^{1 – 3} When activated, Rab proteins partake in crosstalk through shared effector proteins or through Rab activators to ensure vesicle traffic is spatiotemporally regulated. ¹ Homologous to YTP1 in yeast, the Rab1 human proteins play key roles in regulating ER-Golgi and intra-Golgi transport. They exist as two isoforms in humans, Rab1A and Rab1B. While Rab1A and Rab1B share 92% amino acid identity, understanding their specific roles in membrane trafficking is a matter of ongoing investigation. ^{4 – 6} Rab1B has been proposed to function in the initial stages of the secretory pathways, serving to assemble and disassemble machinery required for vesicle fission and fusion, ⁴ whereas Rab1A exhibits unique functions such as its involvement in cell adhesion and migration, and plays a role in facilitating autophagosome formation, an early step in the autophagy pathway ^{7 , 8}

Elevated expression of R AB1A and RAB1B genes have implications in various cancer types, including colorectal cancer, ⁹ hepatocellular cancer, ¹⁰ gliomas, ¹¹ tongue carcinomas, prostate cancer ¹² for RAB1A and colorectal cancer, ¹³ hepatocellular cancer, ¹⁴ and prostate cancer ¹² for RAB1B. Rab1A in human cancer is highly studied in comparison to Rab1B as abnormal expression of Rab1A activates mTORC1 signalling, promoting tumour growth, invasion and ultimately cancer progression. ⁹ Rab1 proteins are also involved in the pathogenesis of Parkinson’s disease, characterized by accumulation of α-synuclein. Inhibition of ER-Golgi traffic has been reported to trigger α-synuclein aggregation, suggesting that an increase in production of Rab1 proteins can potentially rescue this α-synuclein toxic phenotype. ¹⁵ Further research is required to understand the role of Rab1A and Rab1B in various diseased states and their potential as therapeutic targets to slow the progression of cancer and neurodegeneration. In-depth mechanistic investigations would significantly benefit from the accessibility of high-performing antibodies, which can help elucidate the underlying processes and pathways involving Rab1A and Rab1B. An editorial by Biddle et al. can provide valuable insights on how to interpret the antibody characterization data found in this article. ¹⁶

This research is part of a broader collaborative initiative in which academics, funders and commercial antibody manufacturers are working together to address antibody reproducibility issues by characterizing commercial antibodies for human proteins using standardized protocols, and openly sharing the data. ^{17 – 19} Here, twelve commercially available antibodies that target either Rab1A or Rab1B were tested in Western Blot, immunoprecipitation and immunofluorescence applications using a knockout-based validation approach. This article serves as a valuable guide to help researchers select high-quality antibodies for their specific needs, facilitating the biochemical and cellular assessment of Rab1A and Rab1B properties and function.

Our standard protocol involves comparing readouts from wild-type (WT) and knockout (KO) cells. ^{20 , 21} The first step was to identify a cell line(s) that expresses sufficient endogenous levels of a given protein to generate a measurable signal. To this end, we examined the DepMap transcriptomics database to identify all cell lines that express the Rab1 isoforms at levels greater than 2.5 log ₂ (transcripts per million “TPM” + 1), which we have found to be a suitable cut-off (Cancer Dependency Map Portal, RRID:SCR_017655). Commercially available HAP1 cells express Rab1A and Rab1B transcripts at RNA levels above the average range of cancer cells analyzed. Parental and RAB1A and RAB1B KO HAP1 cells were obtained from Horizon Discovery ( Table 1).

For Western Blot experiments, we resolved proteins from WT and RAB1A and RAB1B KO cell extracts and probed them side-by-side with all antibodies in parallel ( Figure 1). ²¹ Figure 1 indicates which antibodies are intended for Rab1A (A) or Rab1B (B). In the results, it was observed that Rab1A antibodies, namely ab302545**, NBP3-11042*, NBP3-11043*, 13075** and 11671-1-AP immunodetected their target, Rab1A protein as a ~23 kDa band in the HAP1 WT lysate while the levels of Rab1A increased by ~2-3 fold in the lysates of Rab1B KO cells. Similarly, Rab1B antibodies 17824-1-AP and PA5-77240 detect Rab1B at ~23 kDa in the HAP1 WT lysate, and revealed a similar ~2-3 fold increase in Rab1B protein level in the Rab1A KO lysate. These results demonstrated that a compensatory mechanism exists to ensure that overall Rab1 protein levels remain balanced.

As per our standard procedure, we next used the antibodies to immunoprecipitateRab1A and Rab1B from HAP1 cell extracts. The performance of each antibody was evaluated by detecting the Rab1A and Rab1B protein in extracts, in the immunodepleted extracts and in the immunoprecipitates using an antibdy that was validated by Western Blot ( Figure 2). ²¹

For immunofluorescence, antibodies were screened using a mosaic strategy, as per our standard procedure. First, the HAP1 WT and RAB1A KO cells were plated together in the same tissue culture wells, using different colour fluorescent dyes to distinguish the two cell lines, and the seven Rab1A antibodies were tested. Then, HAP1 WT and RAB1B KO cells were plated together using the same strategy, and the five Rab1B antibodies were tested. Cells were imaged in the same field of view to reduce staining, imaging and image analysis bias ( Figure 3). Quantification of immunofluorescence intensity hundreds of WT and KO cells was performedfor each antibody tested. The images presented in Figure 3 are representative of the results of this analysis.

In conclusion, we have screened seven Rab1A and Rab1B commercial antibodies by Western Blot, immunoprecipitation and immunofluorescence. Several high-quality antibodies that selectively detect either Rab1A or Rab1B under the standardized experimental conditions were identified in each of the tested applications. In our efforts to address the antibody reliability and reproducibility challenges in scientific research, the authors recommend the antibodies that demonstrated to be underperforming be removed from the commercial antibody market. However, the authors do not engage in result analysis or offer explicit antibody recommendations. A limitation of this study is the use of universal protocols - any conclusions remain relevant within the confines of the experimental setup and cell line used in this study. Our primary aim is to deliver top-tier data to the scientific community, grounded in Open Science principles. This empowers experts to interpret the characterization data independently, enabling them to make informed choices regarding the most suitable antibodies for their specific experimental needs.

The underlying data for this study can be found on Zenodo, an open access repository for which YCharOS has its own community. ^{22 , 23}