Anti gfp monoclonal antibody b 2

Dulbecco’s Modified Eagle’s medium (DMEM), phosphate-buffered saline (PBS), and Hanks’ balanced salt solution were obtained from Nissui (Tokyo, Japan). Fetal bovine serum (FBS) and calf serum (CS) were obtained from JRH Bioscience (Tokyo, Japan). Anti-Myc monoclonal antibody (9E10) was purchased from Millipore (Billerica, MA, USA). Anti-FLAG M2 antibody and anti-α-tubulin antibody (B-5-1-2) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Anti-GFP monoclonal antibody (B-2) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-PI3KAP/XB130 antibody was raised in our laboratory as previously described (12 (link)). Alexa Fluor 488-conjugated anti-mouse IgG antibody was from Invitrogen (Carlsbad, CA, USA). Horseradish peroxidase (HRP)-linked anti-mouse IgG antibody and HRP-linked anti-rabbit IgG antibody were purchased from GE Healthcare (Buckinghamshire, UK). Other chemicals were of reagent grade available commercially.

HEK293 cells were seeded in 6-well plates coated with Poly-l-lysine (PLL; Sigma-Aldrich) at 3.0 × 10⁵ cells/well and incubated for 24 h. The cells were transfected with each expression vector using Lipofectamine LTX with PLUS reagent (Invitrogen) according to the manufacturer’s instructions and then further incubated for 6 h. Then, the medium was replaced with DMEM supplemented with 10% FBS and incubated for another 18 h. The cells were lysed with 1% Triton X-100 containing 1% cOmplete™ Protease Inhibitor Cocktail (Roche). Tissues were homogenized in lysis buffer (20 mM Tris-HCl pH7.4 at 4 °C, 1% Triton X-100, 0.1% SDS, 1% Sodium deoxycholate, 150 mM NaCl) containing 1% protease inhibitor cocktail. Samples were electrophoresed on SDS-polyacrylamide gels, transferred to polyvinylidene difluoride membrane, and probed with the indicated primary antibodies. Secondary antibodies conjugated to horseradish peroxidase were detected using an Immunostar LD/Zeta (FUJIFILM Wako). Anti-GFP monoclonal antibody (B-2, catalog number: sc-9996) was purchased from Santa Cruz Biotechnology. Anti-ABCA1A antiserum was generated against 19 amino acids (EPGKKRRRKKEPLETDLLS) in the ABCA1A linker region between NBD1 and TMD2. The amino acid sequence of the epitope is quite different from ABCA1B and ABCA1C, as shown in Figure S1. The antibody was made at Sigma-Aldrich Japan.