RalA/RalB Antibody

Detection of Human/Mouse/Rat RalA/RalB by Western Blot.

Western blot shows lysates of U937 human histiocytic lymphoma cell line and NIH-3T3 mouse embryonic fibroblast cell line. PVDF membrane was probed with 1 µg/mL Goat Anti-Human/Mouse/Rat RalA/RalB Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3204) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF109). For additional reference, recombinant human RalA and RalB (5 ng/lane) were included. A specific band for RalA/RalB was detected at approximately 25 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

Detection of Human RalA/RalB by Simple Western^TM.

Simple Western shows lysates of Exosome Standards (HT‑29) (NBP3-11685) and U937 human histiocytic lymphoma cell line, loaded at 0.5 mg/ml. A specific band was detected for RalA/RalB at approximately 32 kDa (as indicated) using 10 µg/mL of Goat Anti-Human/Mouse/Rat RalA/RalB Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3204). This experiment was conducted under reducing conditions and using the 2-40kDa separation system.

Detection of RalA/B by Western Blot

Role of RALA and its S194 phosphorylation in regulating RAL activation in MCF7 and MiaPaCa2 cells. (A,B) Western blot detection of RALA (WB: RALA) and actin (WB: actin) in cell lysates of MCF7 and MiaPaCa2 control (CON), RALA KO (KO) and RALA KO cells reconstituted with RALA mutants (WT/S194A/S194D). MCF7 Lysates were further probed for additional proteins of interest, with the results shown in Fig S3C. Blots were reused where required, and the RALA and actin blots from A are also shown for comparison in Fig. S3C. (C,D,E,F) Western blot detection (upper panel) and quantitation (lower panel) of active RALA and active RALB (in samples incubated with GST-Sec5, which pulls down active RALA and RALB from the same lysate), as well as total RALA and total RALB from lysates of control (CON), Cas9 control (Cas9), RALA KO (KO), WT-RALA (WT), S194A-RALA (S194A) and S194D-RALA (S194D) mutant reconstituted MCF7 (C,E) and MiaPaCa2 (D,F) cells. The same GAPDH blot is shown in C and E as the common loading control for RALA and RALB in the MCF lysates, and the same GAPDH blot is shown in D and F as the common loading control for RALA and RALB in the MiaPaCa2 lysates. (G,H) Western blot detection (upper panel) and quantitation (lower panel) of active RALA and total RALA from lysates of MCF7 and MiaPaCa2 cells treated with DMSO (CON), empty nano-vesicle scaffold (DEX), 0.02μM Free MLN and 0.02μM VMLN. The graphs represent the ratio of active to total protein (RALA and RALB) as mean±s.e. from at least three independent experiments. Statistical analysis was done using the Mann–Whitney test, and P-values, if significant, are represented in the graph (*P<0.05, **P<0.01, ***P<0.001). Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/40568758), licensed under a CC-BY license. Not internally tested by R&D Systems.