Anti hsc70

For stimulation, WSU-FSCCL cells were incubated with antibodies and/or DC-SIGN-Fc/HA at 4 °C for 30 min and then rapidly warmed to 37 °C to initiate signaling for the time indicated. Alternatively, FL cells were pre-incubated with DC-SIGN-HA at 4 °C prior to anti-IgM stimulation and rapidly warmed to 37 °C to initiate signaling for 30 s. SDS-PAGE was performed as previously described^{6 (link)} using the following primary antibodies: anti-phospho-ERK1/2, anti-ERK1/2, anti-phospho-AKT^(S473), anti-AKT, anti-phospho-SYK^(S525,526), anti-SYK (all from Cell Signaling Technology), anti-phospho-LYN^(Y396) (Abcam), anti-LYN (Santa Cruz Biotechnologies), anti-HSC70 (Santa Cruz Biotechnologies) and anti-GAPDH (Invitrogen). Images were captured using the ChemiDoc-It Imaging System with a BioChemi HR camera (UVP) and quantified using ImageJ (http://imagej.nih.gov/ij/).

MDM were harvested and lysed on ice in RIPA buffer supplemented with phosphatase inhibitor cocktail 2 and 3 (Sigma-Aldrich) and a cocktail of protein inhibitors (Roche Diagnostic, Meylan, France). Then, cell lysates were sonicated on ice and protein concentration was measured using the Bradford's method. Samples were heated for 5 min at 100°C, loaded in a 4 % stacking gel and then separated by a 8% sodium dodecyl sulfate polymerase gel electrophoresis (SDS-PAGE). Gels were electroblotted overnight onto nitrocellulose membranes. After blocking the membrane with a Tris-buffered saline solution supplemented with 0.1% tween-20 and 5% bovine serum albumin, membranes were hybridized with primary antibodies overnight at 4°C and incubated with appropriate horseradish peroxidase-conjugated (HRP) secondary antibodies. Primary antibodies used were directed against anti-P-MYPT1 (Thr696) (Ozyme SAS, Saint Quentin-en-Yvelines, France) and anti-HSC70 (Santa Cruz Biotechnology, Inc. Heidelberg, Germany). Immunolabeled proteins were finally visualized by chemiluminescence. Full scans of the entire original gels are provided as supplementary material. Densitometry with ImageJ 1.40g software (National Institutes of Health, Bethesda, MD, USA) was used for quantifying intensities of stained bands and normalization to HSC70 content.