Anti total erk1 2

For protein extraction, cells were washed with ice-cold PBS and lysed for 20 min on ice with RIPA (RadioImmunoPrecipitationAssay) buffer (150 mM NaCl, 50 mM Tris pH7.4, 1 % NP40, 1 % sodium-deoxycholat, 1 mM EDTA, 1 mM Na₃VO₄, 25 mM NaF, 1 mM PMSF, 5 mM beta-glycerolphosphat, protease inhibitor cocktail tablets (Complete mini; Roche)). Total cell lysates were cleared by centriguation (10 min, 4 °C, 10,000 g). Protein concentration was measured with the Bradford ProteinAssay (BioRad). Western blots were performed as described in [50 (link)] with the following antibodies: anti-Fibronectin: Santa Cruz, sc-9068; anti-ZO1: Zymed Laboratories, 33–9100; anti-E-Cadherin: BD Transduction Laboratories, 610181; anti-Vimentin: Sigma, V2258; anti-Actin: Sigma, A2066; anti-phospho-AKT (Ser473): Cell Signaling, 9271; anti-total-AKT: Cell Signaling, 9272; anti-phospho-ERK1/2: Sigma, M8159; anti-total-ERK1/2:Sigma,M5670; anti-Pan-Ras: Calbiochem, OP38.
For immunoprecipitation, cells were lysed as described for Western blotting. Equal amounts of protein were incubated with anti-v-H-Ras antibody (Calbiochem, OP01) overnight at 4 °C. Then ProteinA/G plus beads (Santa Cruz) were added and samples were incubated for 1 h at 4 °C. Thereafter, immune complexes were collected by centrifugation, washed twice with ice cold RIPA buffer and subjected to SDS PAGE and Western blotting.

Anti phospho-ERK1, 2 and anti-total ERK1, 2 were purchased from Sigma-Aldrich and anti GAPDH was purchased from Millipore. Goat polyclonal anti-HIV-1 gp120-biotin conjugated antibody was purchased from Abcam (Cambridge, United Kingdom). ATTO 488 Conjugated Goat IgG (H&L) antibody was purchased from Rockland (Gilbertsville, PA, USA). S. aureus LTA was purchased from Sigma-Aldrich and D-galactosamine was purchased from Calbiochem. TNFα and IL-6 ELISA kits were purchased from Biolegend. The plasmid p96ZM651gp160-CD5-opt was obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: from Drs. Yingying Li, Feng Gao, and Beatrice H. Hahn. The plasmid gp41-YFP, provided by Roland Schwarzer encodes a HIV-1 gp41 fusion protein with the c-terminal external parts of the protein replaced by a Yellow Fluorescent Protein.

Whole-cell lysates and immunoprecipitated proteins were subjected to NuPAGE 4%–12% bis-tris acrylamide gel electrophoresis (Life Technologies). Using standard protocols, we probed the blots with the following primary antibodies: anti-phosphotyrosine (Cell Signaling Technology), anti-phosphorylated ERK1/2 (Thr180/Tyr182) (Cell Signaling Technology, #4370), anti-total ERK1/2 (Sigma-Aldrich, M5670), anti-phosphorylated p38 (Thr180/Tyr182) (Cell Signaling Technology, #9205), anti-total p38 (Cell Signaling Technology, #2708), anti-total PCNA (Abcam, ab29-100), and anti-β-actin (Millipore, MAB1501). Proteins were immunodetected with an anti-rabbit IRDye^® 800CW antibody (LI-COR Biosciences, 926-32211) and an anti-mouse IRDye^® 680RD antibody (LI-COR Biosciences, 926-68070). Immunoblots were quantified with ImageStudioLite software (LI-COR Biosciences).