Rabbit anti human bcl 2

ADP, ATP, UDP, and UTP were purchased from Sigma-Aldrich (St. Louis, MO). Rabbit anti-human Bcl-2, total ERK, phospho-ERK antibodies, Rabbit anti-human nucleolin antibody, and ERK inhibitor U0126 were purchased from Cell Signaling Technology (Beverly, MA). P2Y1, 12, 13 agonist 2-MeSADP, P2Y1 selective inhibitor MRS2179, P2Y12 potential inhibitor PSB0739, P2Y13 competitive inhibitor MRS2211 were purchased from Tocris (Bristol, UK). Mammalian expression plasmid pReceive-M29 coding for eGFP-nucleolin fusion protein was purchased from GeneCopoeia (Germantown, MD).

RIPA buffer (Beyotime Biotechnology, Shanghai, China) was used to lyse the cells for total protein extraction. The BCA colorimetric assay (Beyotime Biotechnology, Shanghai, China) was used to quantify the amount of proteins in each sample, and 50 μg of protein were resolved by SDS-PAGE. Rabbit anti-human Bcl-2 (1:500 dilution, Cell Signaling Technology [CST], Danvers, MA, USA), rabbit anti-human cleaved caspase-3 (1:500 dilution, CST), and rabbit anti-human GAPDH (1:500 dilution, CST) were used as primary antibodies followed by HRP-conjugated goat anti-rabbit IgG antibodies (1:1,000 dilution, CST). The ECL reagent (Perkin-Elmer, Waltham, MA, USA) was used to detect immunolabeled proteins. Band intensities were quantified by the AlphaEase FC software (Alpha Innotech, San Leandro, CA, USA).

KKU-100 and KKU-213A cells were plated into 6-well flat-bottom plates at 5 × 10⁴ cells/mL. The cells were treated with PP for 48 h. Cell lysates were extracted with RIPA lysis buffer (150 mM NaCl, 0.5 M Tris-HCl pH 7.4, 1% (v/v) Tween-20, 1% (w/v) sodium deoxycholate, 0.1% (w/v) SDS). Protein concentration was determined using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, California, USA). Protein extracts containing 20 μg protein was solubilized in 4x SDS buffer containing β-mercaptoethanol and boiled at 95°C. Protein were electrophoresed on 10% polyacrylamide gel by SDS-PAGE then transferred to polyvinylidene difluoride membranes. The membranes were incubated in 5% skim milk for 1 h at room temperature and then probed at 4°C overnight with the following antibodies: rabbit anti-human Bcl-2 (Cell Signaling Technology, Massachusetts, US), mouse anti-human Bax (BD Biosciences, San Jose, CA) and mouse-anti-human β-actin (Invitrogen, California, US). β-actin was used as a loading control. After incubation with the respective secondary antibody (Abcam, Cambridge, UK), the band intensity was detected by ECL™ Prime Western Blotting Detection Reagent (GE Healthcare, Illinois, USA) for chemiluminescent detection. The apparent density of the bands on membranes was captured by ImageQuant™ Imager (GE Healthcare, Illinois, US).