Total erk antibody

TH P-1 or HBEpC cells were stimulated as indicated at 37°C in a 5% CO₂ incubator for 1 h. Cells were lysed using the RIPA protein lysis buffer consisting of 10% glycerol, 1% Igepal, 50 mM Tris-pH 7.4, 150 mM NaCl, 1 mM EDTA- pH 8.0, 1% Na-deoxycholate, 0.1% SDS and protease inhibitors (31 (link)). Activation of FAK was evaluated using western blot analysis with antibodies against phospho-FAK (P-FAK) at Tyr 397 (Y397) and Y925 followed by total FAK (Cell Signaling Technology, Danvers, MA, USA). Activation of ERK1/2 was evaluated using western blot analysis with phospho-P44/42 ERK1/2 (Thr202/Tyr204) followed by total ERK antibodies (Cell Signaling Technology). The band intensity was determined using Image J program (NIH) and the ratios were calculated as the intensity of phosphorylated proteins divided by the intensity of total proteins.

Alpha-minimum essential medium (MEM) and fetal bovine serum (FBS) were purchased from Gibco (Carlsbad, CA, USA). Trypsin, phosphate-buffered saline (PBS), and distilled water were purchased from Biowest (Carlsbad, CA, USA). Lipofectamine 3000 reagent was purchased from Invitrogen (Carlsbad, CA, USA). The extracellular signal-regulated kinase (ERK) inhibitor U0126, phospho-ERK antibody, total ERK antibody and peroxidase-labeled secondary antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies for hypoxia-inducible factor (HIF)-1α were purchased from BD Biosciences (Oxford, UK). Antibodies for GAPDH were provided by the Gwangju Institute of Science and Technology, Korea (GIST). The intracellular calcium chelatorBAPTA-AM was purchased from Calbiochem (La Jolla, CA, USA).

Cells were seeded in glass slide when the optimal cell density was achieved and fixed by 3% PFA for half an hour at 4°C. The cells were washed twice with 50mM NH₄Cl and three times with PBS and were then penetrated with PBST (PBS+0.1% Triton X 100) for 15min and blocked with blocking buffer (5% goat serum, 5% fetal bovine serum and 2% bovine serum albumin) for 30min, sequentially. Cells were finally incubated with corresponding primary antibody and then Alexa Fluor conjugated second antibody. The samples were visualized under a confocal microscope. Leg1 antibody [54 ] and BHMT antibody [55 (link)] was generated as described. PDI antibody (Sigma,P7496), Giantin antibody (Abcam, ab24586), PH3 antibody (Santa Cruz, SC-8656-R), Actin antibody (Huabio, R1207-1), GAPDH antibody (Huabio, M1211-1), p-Erk antibody (Cell Signalling Technology, 9101), total Erk antibody (Cell Signalling Technology, 4695), pSmad1/5/8 (Cell Signalling Technology, 9511) antibody were purchased from the companies as indicated.

Transfected cells were treated with 10 nM E2. One hour prior to harvest, cells received a second E2 treatment, and were lysed in RIPA buffer containing protease and phosphatase inhibitors. Cell lysates were then centrifuged at 10 000g for 20 min to remove debris. Protein concentrations were measured using a BCA protein assay kit (Pierce). Proteins separated by SDS–PAGE were transferred onto nitrocellulose membranes. After blocking with blocking buffer (Tris-buffered saline, 0.05% Tween 20, 5% dry skim milk, pH 7.4) for 1 h at room temperature, membranes were incubated for 24 h with phospho-ERK antibody at 1 : 1000 dilution (Cell Signaling Technology, Beverly, MA). After incubation with secondary antibodies conjugated with HRP, proteins were visualized using an ECL detection kit (Pierce) according to the manufacturer's instructions. The membranes were then stripped with a stripping buffer (Pierce) and re-probed with total ERK antibody (Cell Signaling no. 9102). To control for total protein loading, membranes were also probed with an anti-tubulin antibody (Abcam). We used the following catalogue numbers and vendors:

Cultured B cells were washed twice with PBS and homogenized into extraction buffer (8 M urea, 10% glycerol, 1% SDS, 10 mM Tris-HCl pH 6.8, protease inhibitor complete (Roche), 1 mM Sodium-Vanadate). Total cell lysates were resolved on 10% SDS–PAGE and were transferred to nitrocellulose membrane (Bio-Rad). The following primary antibodies were used (Supplementary Table 1): HIF-1α antibody, HIF-2α antibody (Novus), STAT3 phosphorylated at Ser727 and Thr705 antibodies, total STAT3 antibody, phosphorylated ERK antibody, total ERK antibody (Cell Signaling), and β-actin antibody (Sigma). The western blot bands were quantified using ImageJ Software.
The Dynabeads co-immunoprecipitation kit (14321D, Invitrogen) was used for the endogenous co-immunoprecipitation assay and nuclear extracts preparation was described previously^{55 (link)}. Briefly, splenic B cells were enriched from WT mice and stimulated with anti-IgM (10 μg/ml) for 8 h. Five milligrams of nuclear extracts were incubated with 5 μg of anti-HIF-1α antibody (H1α67), anti-pSTAT3⁷²⁷ antibody, or control IgG with Dynabeads protein G according to the manufacturer’s instruction. Immunoblots were then performed using anti-HIF-1α or anti-STAT3 phosphorylated at Ser727 as described above.
Full size images are presented in Supplementary Fig. 11.

For either fish embryo or cultured cells, total protein was extracted using an extraction buffer (63mM Tris-HCl, PH6.8, 10% glycerol, 5% β-Mercaptoethanol, 3.5% SDS) containing 1X Complete (Roche, 11873580001). Western blotting was performed as described previously [27 (link)] using corresponding antibodies as indicated in the figures. Actin antibody (Huabio, R1207-1), GAPDH antibody (Huabio, M1211-1), p-Erk antibody (Cell Signalling Technology, 9101), total Erk antibody (Cell Signalling Technology, 4695), pSmad1/5/8 antibody (Cell Signalling Technology, 9511), Bip antibody (Sigma, G9043), Chop antibody (Sigma, G6916), phosphorylated eIF2a (p-eIF2a) antibody (Cell Signaling Technology, 9721S), and total eIF2a antibody (Cell Signaling Technology, 9720S), and Flag antibody (Sigma, F1804) were purchased from the companies as indicated. Signal intensity of a desired band was calculated by ImageJ software (v.1.48).

Frozen liver tissue was homogenized in RIPA buffer containing protease (P8340; Sigma, St. Louis, MO, USA) and phosphatase inhibitors (201154; Sigma, St. Louis, MO, USA). Crude impurities were removed by brief centrifugation. Fat remaining on the tube wall was also carefully removed in order to prevent contamination of the homogenate. Total protein was separated in 10% SDS-PAGE gels and then transferred to PVDF membranes. The membranes were blocked with TBST containing 5% skim milk and then incubated with total-ERK antibody (1:500; 9102S; Cell Signaling, Danvers, MA, USA), phospho-ERK antibody (1:500; 9106S; Cell Signaling, Danvers, MA, USA), Beclin-1 antibody (Novus Biologicals, Centennial, CO, USA) and LC-3 antibody (Novus Biologicals, Centennial, CO, USA) in TBST containing 1% skim milk overnight at 4 ℃. After washing, the membranes were incubated with horseradish peroxidase-conjugated antibody (1:5000) for 2 h at room temperature. Bands were visualized using enhanced chemiluminescence (Amersham Biosciences, Piscataway, NJ). In each case, GAPDH (1:10,000; ab8245) or α-Tubulin (1:5000; ab7291; Abcam, Cambridge, UK) was detected and used as a loading control. Band intensity was quantitated using ImageLab.