Anti lyn

Anti-ARHGEF5 was generated in rabbits via immunization with GST-mouse or human ARHGEF5 (amino acids 2–204) and affinity purified using a maltose-binding protein-tagged antigen. Anti-Src-pY418, anti-GFP, anti-FAK-pY397, SD208, Alexa Fluor 488 phalloidin, Alexa Fluor 594-conjugated goat anti-rabbit immunoglobulin G, horse radish peroxidase-conjugated goat anti-rabbit immunoglobulin G, anti-mouse immunoglobulin G, anti-occludin and anti-cortactin-pY421 were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Anti-GAPDH, anti-Fyn, anti-Lyn and anti-vimentin were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-v-Src, anti-cortactin (4F11) and anti-phosphotyrosine (4G10) were from Millipore (Billerica, MA, USA). Anti-FLAG (M2) and anti-β-tubulin were from Sigma-Aldrich (St Louis, MO, USA). Anti-E-cadherin, anti-N-cadherin and anti-FAK were from BD Transduction Laboratories (Lexington, KY, USA). anti-Smad2-pS465/467, anti-Smad2, anti-MLC2-pT18/S19, anti-MLC2, anti-Akt and anti-Akt-pS473 were from Cell Signaling Technology Inc. (Beverly, MA, USA). TGF-β1 and TNF-α were from PeproTech (Rocky Hill, NJ, USA). The Akt inhibitor triciribine was from Selleckchem (Houston, TX, USA).

Lysates from peripheral white blood cells and bone marrow cells were prepared in SDS sample buffer and examined by immunoblotting with the following antibodies: anti-phosphotyrosine (4G10, Millipore), anti-phospho-Src(Y416) (D49G4, Cell Signaling Technology), anti-Lyn (Santa Cruz), and anti-GAPDH (Abcam). Horseradish peroxidase-conjugated anti-mouse and anti-rabbit IgG antibodies were obtained from Cell Signaling Technology.

Samples were separated in 10% sodium dodecyl sulfate (SDS)-Laemmli gels and transferred by electroblotting onto nitrocellulose membranes (biostep). Membranes were blocked with dry milk and incubated (overnight) with antibodies detecting phosphorylated/ non-phosphorylated proteins or Hemagglutinin (HA)-tagged TAK1. We used the anti-HA antibody (provided by Prof. Böhmer, Centre of Medical Biomedicine, Jena), anti-pY719-c-Kit/c-Kit, anti-pY705-STAT3/STAT3, anti-pY694-STAT5/STAT5, anti-pS184/pS187-TAK1/TAK1, anti-pS176(IKK1)/pS-177(IKK2) or anti-pY199-IKK2 and anti-IKK1/2, anti-pS32-IκBα/IκBα, anti-pS473-PKB/Akt/PKB/Akt, anti-pY396-Lyn/Lyn, anti-Tubulin and anti-Ubiquitin [Cell Signaling; except anti-c-Kit, anti-TAK1, anti-IKK1/2, anti-Lyn and anti-Ubiquitin (Santa Cruz); anti-pY199-IKK2 and anti-pY396-Lyn (abcam) and anti-Tubulin (Sigma-Aldrich)]. Membranes were washed in 0.1% Tween/TBS and incubated with HRP-conjugated secondary antibodies: anti-rabbit-Ig, anti-goat-Ig (Santa Cruz) and anti-mouse-Ig (Thermo-scientific). Detection was performed using ECL reagent (Pierce).

The following antibodies were used: anti-Src (05–184; Merck, Darmstadt, Germany), anti-Yes (610375; BD Biosciences, San Jose, CA, USA), anti-Lyn (sc-7274; Santa Cruz Biotechnology, Dallas, TX, USA), anti-Csk (610080; BD Biosciences), anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (2275-PC-100; R&D Systems, Minneapolis, MN, USA), anti-p-Tyr (05–1050; Merck), anti-Src family phospho-Y418 (p-SFKs A-loop; ab40660; Abcam, Cambridge, UK), anti-p-Src Y530 (sc-166860; Santa Cruz Biotechnology), anti-p-Lyn Y508 (CSB-PA000691; Cusabio, Wuhan, China)), anti-Fyn (sc-434; Santa Cruz Biotechnology), anti-E-cadherin (#3195; Cell Signaling Technology, Danvers, MA, USA), anti-vimentin (V6630; Sigma-Aldrich), anti-Cbl-b (sc-8006; Santa Cruz Biotechnology) and anti-c-Cbl (sc-1651; Santa Cruz Biotechnology). Horseradish peroxidase-conjugated anti-mouse IgG (#7076; Cell Signaling Technology) and anti-rabbit IgG (711-035-152; Jackson Immunoresearch, West Grove, PA, USA) antibodies were used for Western blotting.

The anti-Lyn, anti-Src, anti-α-tubulin antibodies and horseradish peroxidase conjugated secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-mouse TNF-α, IL-6, and IL-1β ELISA kits were obtained from R&D Systems (Minneapolis, MN). Anti-phospho-tyrosine (4G10) antibody was from Upstate (Temecula, CA), and anti-pLyn (Tyr 396) antibody was purchased from Abcam (Cambridge, MA). Anti-pSrc, Src, pERK, pJNK, p-p38, JNK, p38 and ERK2 antibodies were purchased from Cell Signaling Technology (Danvers, MA). LPS (Lipopolysaccharides from E.Coli 026:B6) was obtained from Sigma-Aldrich Corp. (St. Louis, MO). The LDH cytotoxicity assay kit was from Promega Corporation (Madison, WI). Human Aβ1–42 was purchased from rPeptide (Bogart, GA).

For immunoprecipitation, the cell lysates were incubated on ice with anti-Lyn, anti-PDGFRA anti-JAK2 or anti-IL-5RA antibody (1:100-1:1000 dilutions; Santa Cruz Biotechnology, USA) for 2 h. The immune complexes were collected following incubating with protein A-agarose (Roche, USA) at 4°C for 1 h. The beads were then washed three times with washing buffer and boiled for 5 min in SDS-PAGE sample buffer. The solubilized proteins were separated by SDS-PAGE, transferred to a nitrocellulose membrane (Amersham Biosciences, Sweden), and detected by immunoblotting against antiphosphotyrosine monoclonal antibody, 4G10 (αPY) or Western blotting was performed as described previously [15 (link)]. Blots were probed with the primary antibodies against phospho-Lyn (Y396) (p-Lyn), Lyn, phospho-p85a (PI3K)/(Tyr467)(p-p85a), p85a, phospho-Akt1 (Thr308/Ser473)(p-Akt1) and Akt1 (Santa Cruz Biotechnology, USA), phospho-Stat5(Tyr 694), and Stat5 (Invitrogen, USA), phospho-MAPK p44/42(Thr 202/Tyr 204), MAPK, phospho-JAK2 (Tyr1007/1008)(p-JAK2), JAK2 and β-actin (Cell Signaling Technology, USA) followed by incubation with the secondary antibodies were used peroxidase-conjugated goat antimouse IgG or goat anti-rabbit IgG (Jackson ImmunoResearch Inc., USA) and enhanced chemiluminescent substrate. Densitometry analysis was performed on exposed films using Quantity One v4.62 software (Bio-Rad, USA).