Goat anti rabbit hrp conjugate

Cells were lysed in M‐PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific) supplemented with protease inhibitor cocktail (Thermo Fisher Scientific), 50 mM NaF and 2 mM Na₃VO₄. Protein concentration was evaluated with a BCA Protein Assay Kit (Thermo Fisher Scientific). Western blotting was performed using standard techniques; total cellular protein from the cells were loaded in each lane and separated on SDS‐PAGE. SDS‐PAGE gels were transferred onto PVDF membranes (Immobilon‐P, Millipore). The membranes were blocked with 5% milk in TBST and then incubated with primary antibody, 1:1000 diluted AXL C89E7 rabbit mAb (#8661), 1:500 diluted Vimentin V9 mouse mAb (sc‐6260), 1:500 diluted anti‐pan cytokeratin C11 mouse mAb (sc‐8018, Santa Cruz Biotechnology), or 1:4000 diluted anti‐β‐actin mouse mAb (sc‐47778, Santa Cruz Biotechnology), overnight at 4°C. The membranes were subsequently incubated with secondary antibodies, 1:2000 diluted goat‐anti‐rabbit HRP Conjugate (W401B, Promega) or 1:2000 diluted goat‐anti‐mouse HRP Conjugate (W402B, Promega) for 1 h at room temperature. Immunoblots were developed with Clarity Western ECL Substrate (Bio‐Rad) and visualized with a WSE‐6100 LuminoGraph (ATTO).

For the quantification of proteins in PASMCs, western blots were performed as previously described (Veith et al., 2022 (link)). The following antibodies were used: mouse monoclonal anti-β-actin (1:50,000; Sigma-Aldrich, Cat# A5316; RRID:AB_476743), rabbit polyclonal anti-p38 mitogen-activated protein kinase (MAPK) (1:1000; Cell Signalling Technology, Danvers, MA, United States, Cat# 9212, RRID:AB_330713, antibody was validated by Sato et al. (Sato et al., 2014 (link))), rabbit monoclonal anti-phospho-p38 MAPK (1:1000; Cell Signalling Technology, Danvers, MA, United States, Cat# 4511, RRID:AB 2139682), goat anti-mouse horseradish peroxidase (HRP) conjugate (1:5000; Promega, Mannheim, Germany, Cat# W402B, antibody was validated by Sato et al. (Sato et al., 2014 (link))) and goat anti-rabbit HRP conjugate (1:5000; Promega, Cat# W401B). Logarithmic values of the data were used for the statistical analysis.