Anti p21 waf1 cip1

Total protein extracts were prepared by lysing cells in ice for 30 min in NP40 lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM EGTA, 1 mM EDTA) supplemented with protease and phosphatase inhibitors (5 mM phenylmethylsulfonyl fluoride PMSF, 3 mM NaF, 1 mM DTT, 1 mM NaVO₄).
All protein extracts were quantified by Bradford assay and equal amounts (30 μg) were loaded onto 8% denaturing SDS polyacrylamide gel electrophoresis (SDS-PAGE), transferred for 2 hours to pure nitrocellulose membrane (Trans-Blot Transfer Medium, Biorad, Hercules, CA). Membranes were blocked in 5% milk-TBS-0.05% Tween 20 for 1 hour and incubated overnight with the indicated primary antibodies. The antibodies antiVDR (C2O) (sc-1008, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-p21 waf1/cip1 (#29475, Cell Signalling Technology, Inc. Danvers, MA) and anti-GAPDH (sc-47724, Santa Cruz Biotechnology, Santa Cruz, CA, USA) were diluted in 5% milk-TBS-0.05%. Secondary antibodies were horseradish peroxidase-conjugated (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Signal intensity was quantified using the ECL reagent (Amersham, GE Healthcare, Piscataway, NJ, USA) for the chemo-luminescence detection.

Cell and tissue was homogenized in RIPA lysis buffer (Promega, Madison, US) on ice for 30 min. The concentration of the total protein was measured by the BCA assay (Beyotime, Beijing, China). Western blot analyses were performed as previous study. The antibodies used in this study were as follows: anti-MRPS23 (1:1500; Santa Cruz Biotechnology, Santa Cruz, CA), anti-E-cadherin and anti-Cyt c (1:2000–5000; Abcam, Cambridge, UK), anti-GAPDH, anti-p53, anti-p21^WAF1/CIP1, and anti-vimentin (all 1:1000; Cell Signaling Technology).

Cells were lysed in protein extraction buffer (50 mM HEPES, 5 mM EDTA, 50 mM NaCl, 1% Triton X-100, 50 mM NaF, 10 mM Na₂P₂O₇, 1 mM Na₃VO₄, 5 μg/mL aprotinin, 5 μg/mL leupeptin, 1 mM PMSF, and protease inhibitor cocktail). Lysates containing equal amounts of proteins were separated by SDS–PAGE and transferred onto polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA). The blots were blocked with a 5% skim milk solution and incubated with the following antibodies: anti-Dicer, anti-cyclin A, anti-cyclin D1, anti-cyclin E, anti-p21^WAF1/CIP1, anti-EZH2,(Cell Signaling Technology, Danvers, MA), anti-HDAC2, anti-GAPDH and anti-CDK2 (Santa Cruz Biotechnology, Santa Cruz, CA), anti-N-cadherin, anti-E-cadherin, anti-vimentin and anti-fibronectin (BD Transduction, San Jose, CA), anti-DNMT1 (Abcam, Cambridge, MA) and anti-H3K27me3 (Millipore, Billerica, MA). The Immobilon™ Western blot detection system (Millipore, Billerica MA) was used to detect bound antibodies. The intensities of the Western blot bands were quantified using LAS 3000 (Fuji Photo Film Co., Japan).

Proteins were resolved by SDS-PAGE and transferred onto nitrocellulose membranes (10600001, GE Healthcare, Piscataway, NJ, USA). The membranes were then probed with the appropriate dilutions of anti-DRG2 (#14743-1-AP, Proteintech), anti-β-actin (A5441, Sigma), anti-p53 (DO-1, sc-126, Santa Cruz), anti-phospho-p53 (Ser15, #9284), anti-caspase-3 (#9662), anti-PARP (46D11, #9532), anti-phospho-histone H2A.X (Ser139, #2577), anti-p38 MAPK (#9212), anti-phospho-p38 MAPK (Thr180/Tyr182, #9211), anti-p21 Waf1/Cip1 (DCS60, #2946), and anti-α-Tubulin (#2144, Cell Signaling). Immunoreactivity was detected using Amersham ECL Prime (RPN2232, Cytiva). Membranes were exposed at multiple time points to ensure that the images were not saturated. If required, the band densities were analyzed with NIH image software and normalized by comparison with the densities of internal control β-actin bands.

Cells and tissues were lysed with cold RIPA buffer (50 mM Tris-Cl, PH 8.0, 2 mM EDTA, 150 mM NaCl, 1% NP-40, 0.5% Na-Deoxycholate, 2% SDS, 10 mM NaF) supplemented with mixture of protease inhibitors (Sigma) and phosphatase inhibitors (Sigma). For the tissue samples, sonication was additionally applied. The concentration of total protein was measured using BCA protein assay kit (Thermo Scientific Pierce, Rockford, IL). For the soluble protein detection, the conditioned media was collected and centrifuged at 13,000 rpm for 20 min before collecting the supernatant. Proteins were resolved by SDS PAGE and electrotransferred to PVDF membrane. Primary antibodies used in immunoblot analyses are as follows: anti-FCN3 (R&D systems; AF-2367), anti-V5 (Invitrogen; R96025), anti-p53 (Santa Cruz Biotechnology, Santa Cruz, CA; sc-126), anti-p21 Waf1/Cip1 (Cell Signaling Technology, Beverly, MA; 2947), anti-CDC25C (Cell Signaling Technology; 4688), anti-Cyclin B1 (Cell Signaling Technology; 4138), anti-Cyclin D1 (Cell Signaling Technology; 2926) anti-PARP (Cell signaling Technology; 9542), anti-HSPA5(Abcam; ab21685), anti-DDIT3 (Cell Signaling Technology; 2895), and anti-α-Tubulin (Sigma; SAB3501072). After applying appropriate secondary antibodies, proteins were detected by enhanced chemiluminescence detection kit (AbClon, Korea) and ChemiDoc™ Imaging system (Bio-Rad)

Lysates from breast cancer cell lines (50 µg of protein extracts) were analyzed by Western blot to check the levels of protein expression. The following antibodies were used: anti KCTD15 (GTX50002, GeneTex Cat# GTX50002), anti-p27 Kip1 (D69C12, Cell Signaling Technology Cat# 13715), anti-p21 Waf1/Cip1 (Cell Signaling Technology Cat# 2947), anti β-actin (Abcam Cat# ab11004) and anti β-tubulin (Sigma-Aldrich Cat# T0198 as internal controls). Proteins were acquired using the ChemiDoc Imaging System (Bio-Rad, Hercules, CA, USA) coupled with Image Lab software (Bio-Rad, Hercules, CA, USA).