Anti rabbit igg antibody

Expression of the enzymes involved in glucose metabolism and in cytosolic and ER PPP was determined by Western blot, using standard procedures. Anterior and posterior brain regions were homogenized in PBS in the presence of a protease inhibitor cocktail for mammalian cells, and total protein was measured by Bradford assay. After SDS-PAGE, performed according to the standard method on 10% precast gels (BioRad), proteins were transferred to a nitrocellulose membrane. The membrane was blocked for 1 h with Tris Buffered Saline (TBS) plus 0.15% Tween 20 (TBSt) containing 5% nonfat dry milk and incubated overnight at 4 °C with the following rabbit polyclonal antibodies: anti-HK II (1:1000, Cell Signalling #2867), anti-G6Pase (1:1000, Abcam, ab93857), anti-PFKP (1:200, Cell Signalling #8164), anti-G6PD (1:1000, Abcam, ab210702), anti_H6PD (1:1000, Abcam, ab170895), or anti-actin (1:10,000, Thermo-Fisher MA5-11869). After washing with TBSt, the membrane was incubated with an anti-rabbit IgG antibody conjugated with horseradish peroxidase (HRP) (BioRad) and developed with Clarity Western ECL Substrate (BioRad). Bands were detected and analyzed for density using an enhanced chemiluminescence system (Alliance 6.7 WL 20 M, UVITEC, Cambridge, UK) and UV1D software (UVITEC). Bands of interest were normalized for actin levels in the same membrane.

RIPA Lysis Buffer (Thermo Fisher Scientific) was used for total protein extraction from chondrocytes, and the extracted proteins were separated using SDS-PAGE and transferred to PVDF membranes, which were incubated in 5% milk for 2 h at room temperature to block nonspecific binding. After washing thrice with PBS buffer, the membranes were incubated (overnight at 4°C) with primary antibodies against autophagy-related gene (ATG) 7, LC3-II/LC3-I, PI3K, p-AKT1, AKT1, p-mTOR, mTOR, p70S6K, p-p70S6K, or GAPDH; all primary antibodies were from Cell Signaling Technology (CST; Boston, MA, USA) and were used at 1:1000 dilution. Next, the membranes were incubated with anti-rabbit IgG antibody (1:1000, CST) for 2 h at room temperature, and then the immunoreactive proteins on the membranes were detected using an imaging system (Bio-Rad, Hercules, CA, USA) [23 (link)].

Anti-CFTR, mouse monoclonal antibodies (clone 24–1 (ATCC) and MM13-4 (Millipore)) were used as described previously [23 (link)]. Mouse anti-AP50/μ2 monoclonal antibody was purchased from BD Transduction Laboratories and used in 1:500 dilutions for immunoblotting. Rabbit polyclonal antibody to mannose-6-phosphate receptor, c-Cbl and Nedd4-2 were purchased from Abcam. Rabbit polyclonal antibody against Dab2 was from Santa Cruz Biotechnology and used in 1:200 dilution. Rabbit polyclonal, anti-CHIP antibody was from Thermo Scientific. Polyclonal anti-β-actin antibody was purchased from Sigma-Aldrich. Alexa Fluor 488-labeled goat anti-mouse IgG antibody and Alexa Fluor 594-labeled goat anti-rabbit IgG antibody were from Invitrogen and used in 1:200 dilution. HRP-conjugated goat anti-mouse IgG antibody and anti-rabbit IgG antibody were from Bio-Rad Laboratories. The SuperSignal West Pico chemiluminescence substrate was purchased from Pierce Chemical Co. The ΔF508 CFTR corrector VX-809 was purchased from Selleck Chemicals. All other chemicals were from Sigma-Aldrich or Thermo Scientific.

Cells were treated with or without γ-IFN for 3 days, and cell lysates were prepared from the treated cells. Cells were transiently transfected with the mouse GILT expression plasmid or HIV-1-based vector construction plasmids, and cell lysates were prepared from the transfected cells 2 days after the transfection. Cells were treated with or without concanamycin A (CMA) (1 nM) for 1 day, and then cell lysates were prepared from the treated cells.
The protein concentrations of cell lysates were measured using a microBCA assay. Equal protein amounts were subjected to SDS-PAGE. The protein amounts were determined based on a cell lysate with the lowest protein concentration. Proteins separated by SDS-PAGE were transferred onto PVDF membranes. These membranes were then treated with goat anti-GILT (Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-LC3, anti-IDO1 (Santa Cruz Biotechnology), or mouse anti-actin (Santa Cruz Biotechnology) antibodies. Then, the membranes were treated with HPR-conjugated anti-mouse IgG antibody, anti-rabbit IgG antibody, or protein G (Bio-Rad, Chicago, IL, USA). Antibody-bound proteins were visualized using ECL reagent (Bio-Rad).

Western blot was performed for PVH protein detection. Fifty micrograms of PVH protein was separated by SDS and blotted onto PVDF membranes. After the membrane was blocked with 5% skim milk at room temperature for 2.5 h, it was incubated overnight with anti-Angptl2 (1:1000, Cat No. 12316–1-AP), anti-Erbb2 (1:1000, Cat No. 18299–1-AP), anti-Klotho (1:1000, Cat No.28100–1-AP), anti-Ednra (1:1000, Cat No. Ag24788), anti-Ccr5 (1:1000, Cat No. 17476–1-AP), anti-Gnb3 (1:1000, Cat No. 10081–1-AP), anti-Gpr81 (1:1000, Cat No. 20146–1-AP), anti-Cyp1b1 (1:1000, Cat No. 18505–1-AP) or anti-β-actin (1:1000, Cat No. 20536–1-AP). After washing with TBST, the membrane was incubated with anti-rabbit IgG antibody (1:10,000, Cat No. SA00002-2) for 3 h. Finally, protein was analysed by chemiluminescence and quantitative analysis using ImageLab software (Bio-Rad). All antibodies were purchased from Proteintech (Proteintech Group, Inc, USA).