Anti dhfr

Cells were treated with HQGGT, HQ, GG, SM, BS, GC, BI, BE, WO, and WS for 48 h and processed for immunoblot analysis as previously described [12 (link)]. The following primary antibodies (obtained from Cell Signaling Inc. unless noted) were used at the indicated dilutions: anti-TS, 1:2000 (#9045); anti-E2F1, 1:1000 (#3742); anti-GAPDH, 1:10,000 (#5174; #sc-47724, Santa Cruz); anti-p-NDRG1, 1:1,000 (#5482); anti-NDRG1, 1:1,000 (#9408); anti-p-P65, 1:5,000 (#3033); anti-P65, 1:5,000 (#8242); anti-PUMA, 1:1,000 (#4976); anti-p-RB, 1:1,000 (#9307); anti-RB, 1:1,000 (#9313); anti-CHK1, 1:1,000 (#2345); anti-HSP27, 1:1,000 (#2402); anti-CyclinB1, 1:1,000 (#12231); anti-STAT3, 1:5,000 (#9139); anti-MCL1, 1:1,000 (#5453); anti-CDK4, 1:1,000 (#12790); anti-CDK6, 1:1,000 (#3136); anti-CyclinD1, 1:1,000 (#2978); anti-DHFR, 1:1,000 (#610696, BD Biosciences); anti-TK1, 1:1,000 (#40688, Novus Biologicals). Proteins were detected using SuperSignal West Pico substrate (Pierce; Rockford, IL). Quantitation of signal intensities was performed by densitometry on a HP scanner using NIH IMAGEJ software.

Dimethyl sulphoxide (DMSO), molecular biology grade, used as a solvent for all stocks of chemical agents, was obtained from Roth (Karlsruhe, Germany). All reagents used in flow cytometry analysis were purchased from BD Biosciences Pharmingen (San Diego, CA, United States). The following primary antibodies were used: anti-GAPDH (Merck Millipore, #MAB374, 1:20000, 30 min, RT), anti-p-p65 (Ser529) (Biorbyt, # orb 14916, 1:500, overnight, +4°C), anti-DHFR (BD Biosciences), and anti-TS (Merck Millipore, #MAB4130, 1:500, overnight, +4°C). Secondary goat anti-rabbit IgG-HRP (Dako, #P0448, 1:2000, 1h, RT) and anti-mouse IgG-HRP (Dako, #P0447, 1:1000, 1h, RT) were used. Hoechst 33342 (Life Technologies, #H3569) and anti-mouse Alexa Fluor 555 were used in the IF study. Protease inhibitors (#11 836 153 001) were from Roche Applied Science (Mannheim Germany). The nitrocellulose membrane was from GE Healthcare Life Sciences (Freiburg, Germany), solvents for HRP reaction (Western Bright Peroxide and Western Bright Quantum) were purchased from Advansta, ECL reagent was from Millipore (United States), and CX-4945 was obtained from Biorbyt. Other solvents, reagents, and chemicals were purchased from POCH (Avantor Performance Materials, Gliwice, Poland), Merck, and Sigma-Aldrich Chemical Company (St. Louis, MO, United States).