Anti phospho erk1 2

The following antibodies were used: anti-phospho ERK1/2 (Sigma St Louis, MO), anti-phospho AKT, anti-AKT, anti-EGFR (Cell Signaling, Cambridge, UK,), anti-ERK1/2 (Santa Cruz Biotechnology, Santa Cruz, CA), anti-TERF2 (Novus bio, Cambridge, UK) and α-tubulin (Fischer scientific, Illkirch France).

The following siRNA were from Dharmacon, GE Healthcare; siRNA 1; 5′-CGUGGAGUUUGUAC GGAAA-3′; siRNA 2- 5′-UGACCUAUGCAGUUC GAAA-3′; siRNA 3: 5′-UCUCGAAGCCGUAGUCG UA-3′; siRNA 4: 5′-CAGGAUUCAUGGAGCGGG A-3′. The following 2′O-methyl RNA were from Eurofins Genomics; UTR1: 5′-CUUCCUCCUGCCCGG-3′; UTR3: 5′-GAAACCCUGAGGGAG-3′. The following antibodies were used for immunoblotting and /or immunohistochemistry: anti-SRSF1 (Invitrogen); anti- α-tubulin (Fischer scientific); anti-ERK1/2 (Santa Cruz Biotechnology); anti-phospho ERK1/2 (Sigma). Cells were transfected with siRNA (0.2 μmol/L) or 2′O-methyl RNA (1 μmol/L) by using oligofectamine (Thermo Fischer Scientific).

The following antibodies were used for Western blotting: anti-phospho ERK 1,2 (Sigma St Louis, MO, Reference M8159)), anti-phospho Akt, anti-Akt and anti-phospho S6 Kinase (Cell Signaling, Cambridge, UK, respectively References 4051, 9272, 9234) and anti-TFE3 anti ERKs (Santa Cruz Biotechnology, Santa Cruz, CA references sc 5958 (TFE3) and sc 93 (ERK)).

The primary antibodies used were obtained from the following companies: anti-alpha-fetoprotein Ab-2 (1:100 for immunohistochemistry, 1:1000 for Western blotting; RB-365) from Thermo Fisher Scientific, anti-EGF receptor (1:1000, 610017) from BD Biosciences, anti-actin (1:10000, A2066) and anti-phospho-ERK1/2 (1:1000, 05-797R) from Sigma-Aldrich, anti-ERK1/2 (1:1000, 9102) from Cell Signaling Technology, anti-VEGF Ab-1 (1:1000, RB-222) from NeoMarkers, and anti-PECAM-1 (1:250, sc-1506) from Santa Cruz Biotechnology. The secondary antibodies used were Cy3-conjugated goat anti-rabbit IgG (1:500, 111-165-003) from Jackson ImmunoResearch, Alexa Fluor 633–conjugated donkey anti-goat IgG (1:1000, A21082) from Invitrogen, Cy3-labeled goat anti-mouse IgG (1:500, 115-165-003) from Jackson ImmunoResearch, HRP-conjugated goat anti-mouse IgG (1:1000, AP124P) from Millipore, HRP-conjugated goat anti-rabbit IgG (1:1000, 111-035-144) from Jackson ImmunoResearch, and HRP-conjugated goat anti-rat IgG (1:1000) from Chemicon.

The reagents used in this study include 5(S), 6(R)-Lipoxin A₄ (Cayman Chemical, Ann Arbor, MI, USA), N-acetyl-l-cysteine (NAC) (Sigma-Aldrich, MO, USA), and FR180204 (Sigma-Aldrich). The following antibodies were purchased from Bioworld (St. Louis Park, MN, USA): anti-MMP-9, anti-MMP-2, anti-ERK1/2, anti-phospho-ERK1/2; an anti-β-actin antibody was obtained from Sigma-Aldrich.

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.