Hrp conjugated anti rabbit igg

Six hundred thousand cells/well were plated in 6-well plates, 24 hours prior drug treatment. Following drug treatments whole cell lysates were prepared of adherent and floating cells with RIPA lysis buffer containing 1X EDTA-free protease inhibitor cocktail (Roche) and 2X PhosphoSTOP (Roche). Dosed protein samples were separated on pre-cast 4–12 % Bis-Tris gels (Invitrogen) and transferred to nitrocellulose using the iBlot dry blotting system (Invitrogen). Blocked membranes were incubated with primary antibodies against TFEB (#101532; Santa Cruz), LAMP1 (# H4A3-s; Hybridoma Bank), LC3 (#2775; Cell Signaling), 4E-BP1 (#9452; Cell Signaling), phospho-4E-BP1 (#9459S; Cell Signaling), p70-S6K1 rabbit IgG (#9202S; Cell Signaling), phospho-p70-S6K1 (#9205S; Cell Signaling) and GAPDH (#25778; Santa Cruz). HRP-conjugated anti-rabbit IgG (#213110-01; GeneTex) and anti-mouse IgG (#213111-01; GeneTex) antibodies were used as secondary antibodies. For immunodetection membranes were incubated with peroxide and luminol solution (1:1) and analyzed with a chemiluminescence imager (Intas). Protein bands were quantified using the gel analysis tool of ImageJ and normalized to the loading control GAPDH. Blots shown are representative of at least three independent experiments.

Syk inhibitors SykI and BAY 61-3606, PI3K inhibitor LY294002, ERK inhibitor U0126, JNK inhibitor SP600125, and p38 inhibitor SB203580 were purchased from Calbiochem-Merck (Darmstadt, Germany). Raf-1 inhibitor GW5074, methyl-β-cyclodextrin (MβCD), and water-soluble cholesterol were obtained from Sigma-Aldrich.
Antibodies against phospho (p)-Zap-70 (Tyr319)/Syk (Tyr352), p-Akt (Tyr308), p-c-Raf (Ser338), p-ERK1/2 (Thr202/Tyr204), p-JNK (Thr183/Tyr185), JNK, p-p38 (Thr180/Tyr182), p-IKKα/β (Ser176/180), p-NF-κBp65 (Ser536), p-IκBα (Ser32), IκBα, p-c-Jun (Ser63), c-Jun, p-c-Fos (Ser32), and c-Fos were purchased from Cell Signaling (Beverly, MA, USA). Anti-Syk, anti-β-actin, HRP-conjugated anti-rabbit IgG, and HRP-conjugated anti-mouse IgG antibodies were purchased from GeneTex Inc. (Irvine, CA, USA). Blocking antibodies against CR3 (clone 5C6) and Dectin-1 (clone 2A11) were purchased from Serotec (Oxford, UK). Antibodies for cell surface staining, anti-CD11b (clone M1/70), anti-CD18 (clone GAME-46), and APC-conjugated anti-F4/80 antibody were obtained from eBioscience (San Diego, CA, USA), and anti-Dectin-1 (clone 218820) was purchased from R&D Systems (Minneapolis, MN, USA).