Anti gapdh antibody

Anti-LC3B, anti-Bcl-xL, anti-Atg16L1, anti-Rac1, anti-Rho1, anti-Cdc42, anti-phospho-ERK, anti-EEA1 and anti-LAMP1 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Mouse monoclonal antibodies reactive with LRP1 (11H4) were a kind gift from Dr. Strickland, University of Maryland School of Medicine, Baltimore. Fibronectin (EP5) and ubiquitin (P4D1) were from Santa Cruz Biotechnologies (Santa Cruz, CA, USA); anti-Cx43, anti-ERK, anti-Bcl-2 and anti-RPTPβ antibodies were from BD Biosciences (Tokyo, Japan); anti-multi ubiquitin monoclonal antibody (FK1) was from MBL (Nagoya, Japan); anti-GAPDH antibody was from GeneTex (Irvine, CA, USA) and anti-LC3 (clone 1703) antibody was from Cosmo Bio (Tokyo, Japan). Anti-RPTPα rabbit polyclonal antibodies for immunoblotting were provided by Dr. Jan Sap; anti-α-tubulin and anti-FLAG M2 antibodies, N-acetyl-l-cysteine (NAC) and ammonium chloride were from Sigma Aldrich (St. Louis, MO, USA); ProLong Gold Antifade Reagent with DAPI and DIDS were from Invitrogen (Eugene, OR, USA); an ERK inhibitor, U0126, was from Cayman Chemical (Ann Arbor, MI, USA); Rac1 inhibitor, NSC23766 was from Wako Pure Chemical Industries (Osaka, Japan); and GSH was from Merck (Darmstadt, Germany).

Three hundred thousand cells of RAW264.7 were cultured in complete RPMI 1640 medium at 37°C. The cells were infected with 1.5 × 10⁷ cells of L. donovani and incubated for 24h at 37°C. After washing three times with PBS, the macrophages were lysed in RIPA Buffer (50 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with protease inhibitor cocktail (Sigma Aldrich). Cell lysates were diluted with SDS sample buffer, boiled for 5 min and separated by electrophoresis on an SDS-containing 4–20% Tris-HCl gradient gel (Thermo), then transferred to a polyvinylidene difluoride membrane (GE Healthcare Bio-Sciences, USA). After blocking with Block Ace (Sumitomo Dainippon Pharma, Japan), the membrane was probed with anti-SIRPα antibody (RayBiotech, USA; 1:2,000 dilution) and anti-GAPDH antibody (GeneTex, USA; 1:2,500 dilution) diluted with PBS containing 0.05% Tween 20 (PBS-T) plus 10% Block Ace. After washing the membrane with PBS-T three times, it was probed with horseradish peroxidase (HRP)-linked donkey anti-rabbit IgG antibody (GE Healthcare) at 1:5,000 dilution with PBS-T containing 10% Block Ace. Bands were visualized by an enhanced chemiluminescence detection system (GE Healthcare) and analyzed by LAS-3000 mini (Fujifilm, Japan). Densitometric analysis was performed using Image J software from the National Institute of Health.

Cell lysis, immunoblotting, and immunoprecipitation were carried out as previously described [38 (link), 39 (link)]. For immunoprecipitation, cells were lysed with RIPA buffer for 30 min on ice. Alpha-tubulin or FLAG-tagged PIP4KIIγ was immunoprecipitated with specific antibodies. Immunoprecipitated complexes were then subjected to immunoblot analysis. Specific proteins were immunoprecipitated or detected using antibodies against MCAK (Cytoskeleton, Denver, CO), PLK1 (Invitrogen, Grand Island, NY), and FLAG, phospho-serine, α-tubulin, and γ-tubulin (Sigma, St. Louis, MO). Beta-actin or GAPDH was detected with anti-β-actin antibody (Chemicon, Temecula, CA) or anti-GAPDH antibody (Genetex, Hsinchu, Taiwan), respectively, for use as loading controls.