Sc 1681

Freshly obtained skin samples from mice back with hair removal were prepared in a lysis buffer containing 1% Triton X-100, 1% deoxycholic acid, 2mM CaCl₂ and protease inhibitors (10 μg/ml leupeptin, 10 μg/ml aprotinin, 1.8mg/ml iodoacetamide and 1mmol/l phenylmethyl sulfonyl fluoride) and quantified with a BCA protein assay kit (Pierce). Equal amounts of total protein were subjected to electrophoresis on 12% Bis-Tris gels, transblotted onto nitrocellulose membranes and probed with different primary antibodies: Anti-p53 (1:1000, sc-393031,Santa Cruz), anti-p16 (1:750, sc-1681,Santa Cruz), anti-p21 (1:250, sc-397,Santa Cruz),anti-GAPDH (1:1000, HC301-01, Transgen Biotech), anti-Akt (phosphoSer473, 1:5000, GTX28932,GeneTex), anti-Akt (Akt 1+2+3)(1:1500, GTX121937,GeneTex), anti-NF-κB (1:500, sc-8008,Santa Cruz), anti-NF-κB (phosphor Ser536,1:1000,93H1, Cell Signaling Technology), anti-Pten (phosphoThr366/pS370, 1:2000, GTX54620, GeneTex), anti-Pten (1:1000, 138G6,Cell Signaling Technology) respectively, followed by a peroxidase-conjugated secondary antibody (KPL). Immunoreactive bands were detected using ECL kit according to the manufacturer’s instructions. Subsequent reprobing using anti-GAPDH was performed for internal loading control.

Freshly isolated or cultured vessel segments were prepared for immunoprecipitation, one‐dimensional protein gel electrophoresis and immunoblotting as described previously (Shi et al. 2017a). The primary antibodies used were: mouse anti‐PKCε (dilution 1:500; sc‐1681; Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti‐PKCθ (dilution 1:500; sc‐1680; Santa Cruz Biotechnology), mouse anti‐PKCη (dilution 1:500; sc‐136 036; Santa Cruz Biotechnology) and rabbit anti‐PKCδ (dilution 1:1000; ab182126; Abcam, Cambridge, MA, USA). Rabbit anti‐TRPC1 antibody (1 μg mL⁻¹) was generated by GenScript (Piscataway, NJ, USA) using peptide sequences from a previously characterized putative extracellular region (Xu & Beech, 2001). Visualization were performed using anti‐rabbit and anti‐mouse secondary antibodies conjugated to IRDye 800RD or IRDye 680CW (dilution 1:10 000; Li‐Cor Biosciences, Cambridge, UK) as appropriate and the Odyssey Infrared Imaging System (Li‐Cor Biosciences). Protein band intensities were measured using Image Studio software (Li‐Cor Biosciences) and normalized to smooth muscle actin (dilution 1:2000; #ab5694; Abcam) when quantified.

The IHC and TUNEL staining of FFPE tissue and its scoring system were performed, as described previously.^{4 (link)} The mouse primary antibodies used were as follows: PRKCE (1:200, sc-1681, Santa Cruz), MDR1/P-gp (1:200, sc-55510, Santa Cruz).