Anti rabbit igg conjugated to horseradish peroxidase

Whole embryo protein lysates were collected at 2 dpf by homogenizing anesthetized embryos immersed in RIPA Buffer (50 mM Tris, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X 100, protease inhibitor (Roche, cat. no. 11697498001)). 100 mg protein was loaded into individual wells of a Mini-PROTEAN TGX Precast Gel (Bio-Rad) and a western blot was performed as described [59 (link)]. Blots were incubated overnight at 4°C with anti-APOL1 antibody (1:1000; Abcam, EPR2907, ab108315). The membranes were subsequently washed in PBST (0.1% Tween 20) and incubated for 1 hour at room temperature with anti-rabbit IgG conjugated to horseradish peroxidase (1:20,000; GE Healthcare, NA934V). ACTIN antibody (1:1000, Santa-Cruz, cat. no. sc-8432) was used as a loading control.

Skin and keratinocytes were solubilized using lysis buffer [1% NP40, 20 mM Tris-HCl (pH 7.5), 0.15 M NaCl, 1 mM EDTA, plus phosphatase cocktails and protease inhibitor cocktails (Nacalai Tesque)]. Each sample was resolved using 10% SDS-polyacrylamide gels, transferred to PVDF membranes, and incubated overnight with anti-phospho-EGFR (Tyr1068) (D7A5) XP^® rabbit monoclonal antibody (dilution ratio 1:1,000) (#3777, Cell Signaling), anti-EGFR (D38B1) XP® rabbit monoclonal antibody (dilution ratio 1:1,000) (#4267, Cell Signaling), anti-ERK1/2 (137F5) rabbit monoclonal antibody (dilution ratio 1:1,000) (#4695 S, Cell Signaling), anti-phospho-ERK1/2 (T202/Y204) (20G11) rabbit monoclonal antibody (dilution ratio 1:1,000) (#4376 S, Cell Signaling), anti-STAT3 rabbit polyclonal antibody (dilution ratio 1:1,000) (#9132 S, Cell Signaling), anti-phospho-STAT3 (Y705) rabbit polyclonal antibody (dilution ratio 1:1,000) (#9131 S, Cell Signaling), and anti-filaggrin antibody (dilution ratio 1:1,000) (#GTX37695, GeneTex). The bound antibodies were detected with anti-rabbit IgG conjugated to horseradish peroxidase (GE Healthcare). The full uncropped blot images are shown in Supplementary Fig. 1.

Total cell extracts were prepared from exponentially growing cells and were subjected to electrophoresis in 10% SDS-polyacrylamide gel and then transferred onto a PVDF membrane. The membrane was blocked with 5% skim milk. To detect XPA, APE1, or α-tubulin, the membrane was incubated in 1:100 dilution of anti-XPA monoclonal antibody (ab2352, Abcam), 1:2000 dilution of anti-APE1 monoclonal antibody (ab194, Abcam), or 1:10000 dilution of anti-α-tubulin monoclonal antibody (ab7291, Abcam) overnight. After washing with phosphate-buffered saline containing 0.05% Tween 20, the membrane was incubated with 1:2500 dilution of anti-mouse IgG conjugated to horseradish peroxidase (GE Healthcare Bio-Sciences). To detect OGG1 or DNA polymerase β (Pol β), the membrane was incubated with 1:10000 dilution of anti-OGG1 monoclonal antibody (ab124741, Abcam) or 1:1000 dilution of anti-Pol β polyclonal antibody (ab26343, Abcam) overnight. After washing with phosphate-buffered saline containing 0.05% Tween 20, the membrane was incubated with 1:2500 dilution of anti-rabbit IgG conjugated to horseradish peroxidase (GE Healthcare Bio-Sciences). The proteins were visualized by chemiluminescence using the ECL system (GE Healthcare Bio-Sciences).