Hybond enhanced chemiluminescence ecl transfer membrane

Cells were lysed in RIPA buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate, 1 M EDTA, 1 mM Na₃VO₄, 1 mM NaF, and protease inhibitor cocktail). Protein samples were quantified using the Bio-Rad DC Protein Assay Kit II (Hercules, CA, USA), separated by electrophoresis on an 8%, 10%, or 15% SDS-PAGE gel, and transferred to a Hybond enhanced chemiluminescence (ECL) transfer membrane (Amersham Pharmacia, Piscataway, NJ, USA). The membranes were blocked with 3% nonfat skim milk and probed with primary antibodies for P21, CDK2, SKP2, cleaved caspase-3, cleaved caspase-9, E-cadherin (Cell Signaling Technology, Beverly, MA, USA), cyclin E, cyclin A, B-cell lymphoma 2 (Bcl-2), PARP, AKT, p-AKT, and MMP-9 (Santa Cruz Biotechnologies, Dallas, TX, USA), and β-actin (Sigma Aldrich). The membranes were exposed to horseradish peroxidase-conjugated anti-mouse or rabbit secondary antibodies. Protein expression was examined by using an enhanced chemiluminescence (ECL) system (Amersham Pharmacia).

The Western blotting was performed according to Jung et al.’s paper [13 (link)]. Cells were washed in 1XPBS, lysed in NP-40 buffer, and centrifuged at 13,000 rpm for 20 min at 4 °C. The proteins were measured using the Bio-Rad Protein Assay. Thereafter, 20 μg of proteins were separated using SDS-PAGE (8–12%). Furthermore, they were transferred onto Hybond-enhanced chemiluminescence (ECL) transfer membrane (Amersham Pharmacia, Piscataway, NJ, USA). The membranes were blocked in 1 × PBS + 0.1% Tween 20 solutioncontaining 3% skim milk. The membranes were incubated with various antibodies against CNOT2 (1:1000) (#34214), PARP (1:1000) (#9532), p21 (#2947, Cell signaling Technology Inc., Danvers, MA, USA), p53 (DO-1) (1:1000) (SC-126), HA- (SC-7392, Santa Cruz Biotechnologies, Santa Cruz, CA, USA), MID1IP1 (15764-1-AP, ProteinTech Antibody Group, Chicago, IL, USA), and β-actin (1:3000) (A5316, Sigma Aldrich Co., St. Louis, MO, USA). All primary antibodies were diluted in 1XPBS + 0.1% Tween 20 solution. Secondary antibodies were diluted in 1XPBS + 0.1% Tween 20 solution containing 3% skim milk. Protein expression was detected using an enhanced chemiluminescence system (Amersham Pharmacia, Piscataway, NJ, USA) by film.

PC-3 cells were transfected with miR-135a, miR-1290, miR-200c-3p, miR-374b, miR-3195, and control mimic plasmids (Genolution). Two days after transfection, the cells were lysed in radioimmunoprecipitation buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholic acid, 1 M EDTA, 1 mM Na₃VO₄, 1 mM NaF, and protease inhibitor cocktail). Protein samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and electrotransferred onto a Hybond enhanced chemiluminescence (ECL) transfer membrane (Amersham Pharmacia, Piscataway, NJ, USA). After blocking, the membrane was incubated with the following primary antibodies: LC3-II (1:1000; #2775, Cell Signaling Technology, Danvers, MA, USA), PERK (1:1000; #5683, Cell Signaling Technology), IRE1α (1:1000; #3294, Cell Signaling Technology), 78 kDa glucose-regulated protein (GRP78) (1:500; #sc-376768, Santa Cruz Biotechnology, Santa Cruz, CA, USA), ATF6 (1:1000; #65880, Cell Signaling Technology), Beclin (1:1000, #3738, Cell Signaling Technology), and β-actin (1:5000; #4970, Cell Signaling Technology). After washing, the membrane was incubated with horseradish peroxidase-conjugated secondary anti-mouse or anti-rabbit antibodies (1:5000; AbD Serotec, Kidlington, UK). An ECL system (Amersham Pharmacia) was used to visualize the protein bands.