Sc 81492

Immunoblotting was used for measuring the protein levels of ADM, pro-caspase-3, pro-caspase-9, cleaved caspase-3/9, Bax, Bcl-2, cleaved PARP, Akt, p-Akt, Erk1/2, and p-Erk1/2. All cells were lysed in 1% PMSF-contained RIPA buffer. After extraction, protein samples were separated by loading onto SDS-PAGE gel, followed by transferring to PVDF membranes. After that, membranes were blocked with 2% bovine serum albumin in TBST, followed by an overnight incubation (4 °C) with primary antibodies: ADM (ab69117, Abcam, Cambridge, MA, USA), caspase-3 (19677-1-AP, Proteintech, Wuhan, China), caspase-9 (10380-1-AP, Proteintech), Bax (50599-2-Ig, Proteintech), Bcl-2 (12789-1-AP, Proteintech), cleaved PARP (13371-1-AP, Proteintech), Akt (Y409094, Applied Biological Materials Inc., Richmond, Canada), p-Akt (66444-1-1 g, Proteintech), Erk1/2 (67170-1-Ig, Proteintech), and p-Erk1/2 (sc-81492, Santa Cruz, Dallas, TX, USA). Next, membranes underwent an incubation with HRP-labeled secondary antibody, followed by visualization using ECL Substrates (Millipore, MA, USA). GAPDH served as an internal reference.

Cells in culture were lysed using Complete Lysis-M kit (Roche). The protein concentrations of the lysates were quantified using Coomassie Plus Protein Assay Reagent (Thermo Scientific). 10 ug of protein for each sample were loaded on to 8% SDS-PAGE gel. Separated proteins were transferred to a PVDF membrane (L-08008-001, Advansta) in wet transfer buffer. Membranes were blocked with 5% milk in TBST (0.5%) for 1 hour at room temperature before incubation at +4°C overnight with the following antibodies: HER2 (1:1000, ab8054, Abcam), total AKT (1:1000, sc8312, SantaCruz), phospho-AKT (Ser 473, 1:1000, sc-7985-R, SantaCruz), total ERK2 (1:1000, sc-154, SantaCruz), phosho-ERK 1/2 (Thr 202 / Tyr 204, 1:1000, sc-81492, SantaCruz) and beta-actin (1:1000, 634801, Biolegend) in 3% milk powder-TBST. After incubation with HRP-conjugated secondary antibodies, the protein bands were detected using WesternBright Sirius Kit (K-12043-D20, Advansta).

Western blot was performed to detect ERK 1/2 protein. After treatment with 100 nM CNP for 24 h or 100 μM NAC for 1 h L929 cells were irradiated with UVA (30 J/cm²) followed by 2 h incubation. Next, cells were lysed in lysis buffer (1%) and total protein (20 μg) was separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to 0.22-μm nitrocellulose membranes. The membranes were blocked with 5% albumin diluted in a Tris-buffered saline solution containing 1% Tween-20 (TBST) and then incubated overnight at 4°C in solutions with primary antibodies (1: 50) against Erk1/2 (sc-514302), phospho ERK1/2 (sc-81492), or PCNA (1: 10,000, sc-56) (Santa Cruz Biotechnology, Santa Cruz, CA, United States). The membranes were washed three times with TBST before an incubation for 1 h in solution with anti-mouse secondary antibody HRP-conjugated (1:10,000) (Santa Cruz Biotechnology, Santa Cruz, CA, United States). Proteins were detected by western blotting luminol reagent (Santa Cruz Biotechnology, Santa Cruz, CA, United States) using CCD camera imaging system (ImageQuant LAS 500, GE Healthcare Life Sciences, Uppsala, Sweden). Image-J 1.45S software (Wayne Rasband, National Institutes of Health, Bethesda, MD, United States). Quantitation of the relative amount of p-ERK 1/2 was normalized to the control PCNA.