Anti phospho erk antibody

Whole cell lysates were resolved on 12% SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked with TBS-T containing 10% skimmed milk for 10–15 min and probed with anti-phospho-Akt antibodies (Cell Signaling Technology Cat# 9271L RRID:AB_329826) diluted 1:1,000 in TBS-T containing 5% BSA overnight at +4°C; or with anti-phospho-Erk antibodies (Santa Cruz Biotechnology Cat# sc-7383 RRID:AB_627545) diluted 1:500 in TBS-T containing 3% skimmed milk overnight at +4°C. Then anti-phospho-Akt-treated membrane was incubated in the 1:3,000 solution of secondary anti-rabbit antibodies conjugated with HRP and anti-phospho-Erk-treated—with anti-mouse-HRP-conjugated antibodies diluted in TBS-T containing 3% skimmed milk for 45 min at room temperature. Membranes were washed with TBS-T (4 × 10 min). Stained bands were visualized with ECL reagent or femptoECL reagent (Pierce) using LAS3000 imaging program. To confirm equal loading, membranes were stripped and reprobed with anti-GAPDH antibodies (Millipore Cat# MAB374 RRID:AB_2107445) diluted 1:4,000 in TBS-T containing 3% skimmed milk. To detect anti-GAPDH antibody we used secondary anti-mouse antibodies conjugated with HRP (1:1,000).

Anti-EGFR and anti-phospho-EGFR antibodies were purchased from Cell Signaling Technology (Beverly, MA). Anti-ERK and anti-phospho-ERK antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). An antibody specific to heparin-binding epidermal growth factor-like growth factor (HB-EGF) was from R&D Systems (Minneapolis, MN). Anti-GPR40 antibodies were obtained from Epitomics (Burlingame, CA) and Santa Cruz Biotechnology (Santa Cruz, CA). Fluorescein Lotus tetragonolobus agglutinin (LTA) was from Vector Laboratories (Burlingame, CA). Anti-aquaporin-2 (AQP2) antibody was from Abcam (Cambridge, MA). Anti-Tamm-Horsfall protein (THP) antibody was from AbD Serotec (Raleigh, NC). Anti-calbindin-D28K antibody was from Sigma-Aldrich (St. Louis, MO). EETs were a generous gift from Jorge Capdevila (Vanderbilt University). Heparin-Sepharose CL-6B column was from Amersham Pharmacia Biotech (Piscataway, NJ). AG1478 was purchased from Calbiochem (San Diego, CA). CRM197 and all other chemicals were from Sigma-Aldrich (St. Louis, MO).

Cigarette smoke condensate was purchased from the Tobacco and Health Research Institute (University of Kentucky, Lexington, KY, United States). Anti-ERK and anti-phospho-ERK antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, United States). Anti-Akt and anti-phospho-Akt antibodies were obtained from Cell Signaling Technology (Beverly, MA, United States). Roflumilast, U0126, Wortmannin, and LPS (from E. coli serotype 0111:B4) were purchased from Sigma (St. Louis, MO, United States).