Gapdh antibody

Primary antibodies against AEG-1/MTDH (13860-1-AP), E-cadherin (1702-1), N-cadherin (2447-1), and vimentin (2862-1) were obtained from Epitomics Inc. (Burlingame, CA, USA). Primary antibodies against poly-ADP-ribose polymerase (PARP) (BS70001), PI3K (BS3006), Bax (BS1030), Bcl-2 (BS1031), and C-myc (BS2462) were obtained from Bioworld Technology Inc. (Minneapolis, MN, USA). Primary antibodies against NF-κB-p65 (10745-1-AP), caspase-8 (13423-1-AP), caspase-9 (10380-1-AP), and caspase-3 (19677-1-AP) were obtained from Proteintech (Wuhan, China); Primary antibodies against Lamin A (sc-177452), AKT (sc-8312), p-AKT (sc-7985), ERK (sc-154), and p-ERK (sc-23759) were obtained from Santa Cruz Biotechnology (Santa Cruz Biotechnology Inc., CA). The GAPDH antibody was purchased from Boster (Wuhan, China). The secondary antibodies peroxidase-conjugated AffiniPure Goat Anti-Mouse IgG (H+L) (111-035-003), peroxidase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) (111-035-003), Cy™ 3-conjugated AffiniPure Goat Anti-Rabbit IgG(H+L) (111-165-003), and Fluorescein (FITC)-conjugated AffiniPure Goat Anti-Rabbit IgG(H+L) (111-095-003) were obtained from Jackson Immuno Research Laboratories, Inc. (USA). Temozolomide (85622-93-1) was obtained from Meilun Biology Technology (Dalian, China).

Human breast cancer cells (MCF-7, MDA-MB-468) were cultured in DMEM medium supplemented with 10% fetal bovine serum in a humidified atmosphere containing 5% CO2 at 37°C. Erlotinib, Lapatinib, Doxorubicin, U0126 and AZD6244 were purchased from Selleck Chemicals. Human normal IgG was obtained from Roche (Basel, Basel-Stadt, Switzerland). GAPDH antibody was obtained from Boster Biological Technology (Wuhan, China). MTT was purchased from Sigma-Aldrich (St. Louis, Missouri), and phospho-ERK, ERK antibodies were from Santa Cruz Biotechnology (Indian Gulch, California). All other antibodies were purchased from Cell Signaling Technology (Beverly, Massachusetts). The Annexin V-FITC Apoptosis Detection Kit was from BD Sciences.

Human nasopharyngeal carcinoma cells (CNE2, HONE1) were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum in a humidified atmosphere containing 5% CO2 at 37°C. The approaches to modeled the resistant cell sublines was published before [53 (link)]. Acquired BEZ235 resistance were generated as described previously. Briefly, parental cells were continuously exposed to increasing doses of BEZ235 (from 0.05 μmol/L up to 0.4 μmol/L) for 6 to 9 months.
BEZ235, GSK2334470, JQ-1, decitabine and RG108 were purchased from Selleck Chemicals. Human normal IgG was obtained from Roche (Basel, Basel-Stadt, Switzerland). GAPDH antibody was obtained from Boster Biological Technology (Wuhan, China). MTT was purchased from Sigma-Aldrich (St. Louis, Missouri), and phospho-AKT, phospho-MYC, MYC, GSK3α/β antibodies were from Santa Cruz Biotechnology (Indian Gulch, California). All other antibodies were purchased from Cell Signaling Technology (Beverly, Massachusetts). Matrigel was purchased from BD Sciences. The Annexin V-FITC Apoptosis Detection Kit was from Invitrogen Inc. (Carlsbad, California).

Recombinant mouse IL-1β was purchased from R&D Systems (Minneapolis, MN, United States). Lipofectamine 3000 reagent was obtained from Invitrogen (Waltham, MA, United States). Anti-SHP2, β-catenin, p38, p-p38, extracellular signal-regulated kinase (ERK), p-ERK, c-Jun N-terminal kinase (JNK), p-JNK, p65, p-p65, IκBα, p-IκBα, IκB kinase (IKK)-β and p-IKKα/β antibodies were supplied by Cell Signaling Technology (Beverly, MA, United States). MMP3, MMP13 and p-β-catenin(Y142) antibodies were obtained from Abcam (Cambridge, United Kingdom). p-β-catenin(S33/37/141), COL2A1, and AGGRECAN antibodies were purchased from Abclonal (Wuhan, China). GAPDH antibody was purchased from Boster (Wuhan, China) and secondary antibodies were obtained from the Jackson lab.

Protein was extracted from the rats’ ovaries using RIPA (radioimmunoprecipitation assay) lysis buffer and was quantified by using a BCA protein assay kit (Thermo Fisher, SE253117A). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed with 10% separation glue. Then the protein on the gel was transferred to polyvinylidene difluoride (PVDF) membrane under the condition of constant 300 mA. 5% skimmed milk dissolved in Tris-buffered saline with Tween (TBST) was used to block the PVDF membrane after transmembrane. The primary antibody was incubated overnight at 4 °C, and the secondary antibody was incubated for 1 h at room temperature. After that, an ECL kit was used to determine chemiluminescence.
The antibodies were TGF-Beta1 Antibody (Proteintech, 21898-1-AP), FSHR Antibody (Proteintech, 22665-1-AP), Anti-Smad4 (Abcam, ab40759), JNK (Phospho-Thr183/Thr183/Thr221) Rabbit pAb (ZENBIO, 384799), JNK Antibody (Proteintech, 10023-1-AP), Phospho-Smad2 (Ser467) Rabbit pAb (ZENBIO, 310079), Smad2/3 Rabbit pAb (ZENBIO, 382472), Phospho-Smad3 (Ser423/425) Rabbit pAb (ZENBIO, 382919), Smad3 Rabbit pAb (ZENBIO, 382774), TAK1 (Phospho-Ser439) Rabbit pAb(ZENBIO, 385849), TAK1 (3G1) Mouse mAb (ZENBIO, 200993), TRAF6 Rabbit pAb (ZENBIO, 385965), p38 MAPK (CST, 8690S), Phospho-p38 MAPK (CST, 4631S), GAPDH Antibody (Boster, BA2913), HRP-linked Antibody (CST, 7074).

To measure the protein level of MyoD, the total proteins from cultured SMSCs were extracted using the Total Protein Extraction Kit (BestBio, Shanghai, China) and quantified by employing BCA Protein Quantitation Kit (BestBio, Shanghai, China) according to the manufacturer’s instructions. In brief, with ~20 μg of protein per sample, Western blotting was performed by separating protein on a 12% SAS-PAGE, transferring to a PVDF (Millipore, Burlington, MA, USA), blocking with 5% milk in TBS, and then incubating the membrane sequentially with the primary anti-mouse MyoD (1:1000) (Abcam, Bristol, UK) or MyHC (1:1000) (Minneapolis, MN, USA) and the secondary antibody IgG (Beyotime, Shanghai, China). Eventually, we measured the enhanced chemiluminescence signal (ECL) (Solarbio, Beijing, China) after adding horseradish peroxidase (HRP) (Bio-Rad, CA, USA) and the GAPDH antibody (1:1000, mouse) (Boster, Wuhan, China) as a loading control.