Immun blot pvdf membranes for protein blotting

20–25μg of protein lysate with 4x Laemmli buffer (cat# 1610747; Bio-Rad Laboratories was separated on 4–15 % Mini-PROTEAN TGX Precast Protein Gels (cat# 4561086; Bio-Rad Laboratories) and transferred to Immun-Blot PVDF Membranes for Protein Blotting (cat# 1620177; Bio-Rad Laboratories). Membranes were reversibly stained for total protein with Ponceau S Staining Solution (cat# A40000278; Thermo Scientific) to ensure transfer and equal loading prior to antibody incubation. Blocking and antibody incubations were done using StartingBlock T20 (TBS) Blocking Buffer (cat# 37543; Thermo Scientific). Primary antibodies were used at 1:1000 except for anti-thrombospondin-4 and anti-TIMP-1 which were diluted at 1:100. Secondary antibodies conjugated with horseradish peroxidase were used at 1:2500 (cat# 111035003; Jackson ImmunoResearch Laboratories). Clarity Western ECL Substrate (cat# 1705061; BioRad Laboratories) was applies to membranes and visualized using an iBright 1500 Imaging System (Invitrogen). Pierce Reversible Protein Stain Kit for PVDF Membranes (including MemCode; cat# 24585; Thermo Fisher Scientific) was used to ensure equal loading.

15μg of protein lysate diluted in 4x Laemmli buffer (cat# 1610747; Bio-Rad Laboratories) was separated on 4–15% Mini-PROTEAN TGX Precast Protein Gels (cat# 4561086; Bio-Rad Laboratories) and transferred to Immun-Blot PVDF Membranes for Protein Blotting (cat# 1620177; Bio-Rad Laboratories). Membranes were blocked using StartingBlock T20 (TBS) Blocking Buffer (cat# 37543; Thermo Scientific). Primary antibodies anti-p-SMAD3 (71 ab52903, Abcam) and anti-SMAD3 (71 ab40854, Abcam) were used at 1:1000. Secondary antibodies conjugated with horseradish peroxidase were used at 1:2,500 (cat# 111035003; Jackson ImmunoResearch Laboratories). ECL Substrate (cat# 1705061; BioRad Laboratories) was briefly applied to the membranes and visualized using an iBright 1500 Imaging System (Invitrogen). Pierce Reversible Protein Stain Kit for PVDF Membranes (including MemCode; cat# 24585; Thermo Fisher Scientific) was used to ensure equal loading. Band intensity was quantified using the gel tool in FIJI (NIH).