Anti lyn

Anti Cd11a inhibitory antibodies MHM 24, TS1.22.1.1.13.3 and Tranferrin receptor antibodies G1/221/12 were obtained from the mouse hybridoma bank (DSHB) as cell culture supernatants. Other antibodies and their sources were: anti-M2 FLAG antibody (Sigma), anti-phospho LCK Y394 (Thermo scientific), anti-LYN (Cell Signaling Technologies), anti-HCK (Cell Signaling Technologies), anti-FGR (Cell Signaling Technologies), anti-phospho Src family Y416 (recognizing phosphor-LYN, HCK, and FGR; Cell Signaling Technologies), anti-M2 Flag Beads (Sigma), Glutathione Sepharose (GE healthcare).

The following antibodies were used for immunoblotting: anti-SYK (N-19; Santa Cruz), anti-IgM-HRP (Southern Biotech), anti-Cav1 (D46G3; Cell Signaling), anti-RAC1 (Millipore), anti-LYN (Cell signaling) and anti-Tubulin (Sigma).
The following antibodies were used for flow cytometry: anti-B220-PECy7 (RA3-6B2), anti-IgD (11-26), anti-IgM (polyclonal), anti-CD19 (1D3), anti-CD4 (GK1.5 and RM4-5), anti-CD8 (53-6.7), anti-CD93 (AA4.1), anti-CD69-PECy7 (H1.2F3), anti-CD86-PE (GL-1), anti-GL7, anti-CD95 (15A7) (all from eBioscience), anti-λLC λ1, λ2 & λ3 (R26-46), anti-κLC (187.1), anti-CD45.1 (A20), anti-IgD-FITC (11-26c.2a), anti-CD45.2 (105), Ki-67 (B56) (all from BD Pharmingen), anti-CD19 (6D5) (Biolegend). NP-BSA-biotin was from Biosearch Technologies. Anti-IgM-Fab’2-Alexa 649 was from Jackson ImmunoResearch (goat polyclonal). CSFE was from Sigma.

The following antibodies were used for immunoblotting assays: anti-AKT (#9272), anti-ERK1/2 (#4695), anti-GSK-3B (#9315), anti-MCL-1 (#4572), anti-SYK (#2172) and anti-LYN (#4576) were purchased from Cell Signaling (Danvers, MA, USA). Anti-XIAP (#610762) was purchased from BD Bioscience (San Jose, CA, USA). Anti-HSP90B (#837159A) and anti-GAPDH (#TAB1001) were purchased from Thermo Fisher Scientific (San Jose, CA, USA). Anti-BCL-2 (#k2206) was purchased from Santa Cruz Biotechno-logy (Santa Cruz, CA, USA). Annexin V-fluorescein isothiocyanate and 7-Aminoactinomycin D were obtained from BD Bioscience. The propidium iodide and InnoCyte Flow Cytometric Cytochrome c Release kit were purchased from EMD Millipore (Kankakee, IL, USA). PU-H71 and PU-H71-conjugated agarose beads were obtained from Dr Gabriela Chiosis and were prepared as previously reported.^{17 (link)} Amaxa Solution V Nucleofector kit was purchased from Amaxa (Cologne, Germany). Pools of siRNA against human BTK, AKT and HSP90 were customarily designed and purchased from Thermo Scientific (Waltham, MA, USA).

For cell sample preparation, cells were lysed with an NP-40 buffer with protease inhibitor cocktail (Roche, 11697498001). For tissue sample preparation, single-cell suspensions derived from mouse tumors were incubated with anti-CD11b antibody–conjugated microbeads (1:100; Miltenyi Biotech, 130-049-601) for 15 min at 4°C and separated by MACS column with a separator. The eluted cells were lysed with NP-40 buffer with protease inhibitor cocktail and phosphatase inhibitor (Thermo Fisher Scientific, 78428). Total protein (20 μg) was resolved by 4 to 15% precast SDS–polyacrylamide gel electrophoresis (Bio-Rad) and followed by transfer. Polyvinylidene difluoride membranes were blotted with anti–phosphoSrc (Tyr⁴¹⁶; Cell Signaling Technology, 6943), anti-Hck (1:1000; Cell Signaling Technology, 14643; ABclonal, A14537), anti-Lyn (1:1000; Cell Signaling Technology, 2796), anti-FLAG (1:1000; GenScript, A00187-100), anti–arginase 1 (1:500; Santa Cruz Biotechnology, sc-20150), or anti–glyceraldehyde-3-phosphate dehydrogenase (1:3000; Cell Signaling Technology, 5174) antibody overnight at 4°C. Proteins were detected with horseradish peroxidase–conjugated secondary antibodies (Bio-Rad) and enhanced by enhanced chemiluminescence development (GE Healthcare, RPN2232).