Anti his antibody

Galactose-induced or non-induced yeast cells were collected by centrifugation (5,000 × g, 5 min, 4°C) from 500 μl of yeast culture and resuspended with 100 μl of water and 100 μl of 0.2 M NaOH, followed by incubation at room temperature for 5 min. Cells were then collected and resuspended with 100 μl of 1 × yeast SDS sample buffer (60 mM Tris-Cl pH 6.8, 4% β-mercaptoethanol, 4% SDS, 0.01% bromophenol blue, and 5% glycerol) and boiled for 5 min. Yeast microsomal proteins were also prepared from galactose-induced cells harboring BrF3′H or the empty vector for immunoblot analysis. Ten microliters of total protein and 20 μg of microsomal protein were separated by 10% SDS-PAGE and electro-transferred onto a polyvinylidene fluoride membrane. After being blocked with skim milk in TBS-T buffer (50 mM Tris-Cl pH 7.4, 150 mM NaCl, 0.1% Tween 20, and 5% skim milk), the membrane was incubated with anti-His antibodies (Santa Cruz Biotechnology, Dallas, TX, United States) diluted 1/5000 at 4°C for 16 h. The membrane was washed with TBS-T buffer, and HRP-conjugated secondary antibodies (Thermo Fisher Scientific) diluted 1/5000 were then applied at 4°C for 2 h. Chemiluminescent signal was detected using West-Q Femto clean ECL solution (GenDEPOT) and the ImageQuant™ LAS 4000 system (Fujifilm, Tokyo, Japan).

HEK293T cell line was maintained in DMEM (HIMEDIA, India) and THP-1 cell line (30 (link)) in RPMI-1640 (HIMEDIA, India) media, supplemented with 10% Fetal Bovine Serum (Gibco, USA), 100 U/ml of penicillin, and 100 ug/ml of streptomycin (HIMEDIA, India) at 37°C with 5% CO₂ (31 (link)).
Different antibodies used to monitor the expression and localization of various proteins include anti-Myc antibody (Santacruz biotechnology (sc40); GAPDH (Santacruz biotechnology (sc47724); GFP antibody (Santacruz biotechnology, sc-9996 and ABclonal, AE011); anti-His antibody (Santacruz biotechnology, sc-8036), CD206 antibody (Santacruz biotechnology, sc-58986 PE), CD64 antibody (Santacruz biotechnology, sc-1184 FITC) and secondary antibodies include anti-mouse HRP (cell signaling, 08/2017), anti-rabbit HRP (Santacruz biotechnology, sc-2357) or anti-mouse Alexa 647 (Ab150115). The quantification of HIV for infection assays was performed using HIV-1 p24 Capture ELISA Assay Kit (ABL Inc, USA) and the released HIV antigens were measured using Microlisa HIV Ag & Ab assay kit (detects gp41, gp120, and p24) (JMitra &Co Pvt Ltd, India).

Poly‐His tagged VNI2 (His‐VNI2) was prepared according to a previous report (Yamaguchi, Ohtani, et al., 2010 (link)). ATAF2 cDNA was integrated into the pMAL‐GWRFC. MBP‐ATAF2 was expressed in E. coli strain BL21 trxB (DE3; Cosmo Bio) in the presence of isopropyl ß‐D‐thiogalactoside (IPTG) and purified with amylose resin (New England Biolabs). His‐VNI2 and/or MBP‐ATAF2 proteins were incubated with the amylose resin for 90 min at 4°C. The proteins immobilized with the resin were subjected to immunoblot analysis. His‐VNI2 protein was detected with an anti‐His antibody (Santa Cruz Biotechnology) and an anti‐rabbit IgG antibody (Amersham Biosciences).

Western blotting was conducted with the standard method. Ultimately, the PVDF membranes (Bio-Rad) were incubated with anti-STAT3 antibody (#9139, CST, USA), anti-p-STAT3(#9145, CST, USA), anti-Jak2 antibody (#3230, CST, USA), anti-p-Jak2 antibody (#3771, CST, USA), and tag antibodies, including an anti-HA antibody (Santa Cruz Biotechnology, USA), anti-Flag antibody (F7425, Sigma, USA) and anti-His antibody (Santa Cruz Biotechnology, USA). Asymmetric dimethyl-arginine (ASYM25, anti-Rme2,09-814) was purchased from Millipore (Billerica, MA). An anti-GAPDH antibody from Sigma was used as the internal control.

HK-2 and Caki-1 cells used in this study were obtained from ATCC. Cell culture media and supplements, Dulbecco’s modified Eagle’s medium (DMEM) (Cat: MT10090CV; Corning), Macoy’s 5 A media, fetal Bovine serum (FBS) (F0926; Millipore Sigma), and antibiotic antimycotic solution (Cat: 30–004-Cl; Corning) were used. Primary antibodies used were integrin β1 (D2E5) Rabbit mAb (Cat: 9699; Cell Signaling); antibody for active integrin β1 12G10 (Cat: MAB2247; Millipore Sigma), integrin β1 (Cat: MABT821; Millipore Sigma), integrin β1, (Santa Cruz Biotechnology Cat: SC-374430), ACE2 monoclonal antibody (Cat: MA5–32307; Thermo Fisher), GAPDH (Cat: 5174; Cell Signaling). Anti His antibody (Cat: SC53073, Santa Cruz Biotechnology). Spike protein (Cat: 230–20407–200, Ray Biotech) and the Control protein (Cat: 230–20001, Ray Biotech).

The anti-His antibody (Santa cruz Biotechnology Inc., CA) and HRP-conjugated anti-rabbit secondary antibody (Promega) were used at 1:5,000 and 1:10,000 dilutions, respectively. The primary antibody against PfalMre11 was generated in rabbit and used at 1:4,000 dilutions, and HRP-conjugated anti-rabbit secondary antibody (Promega) was used at 1:12,000 dilutions. As a control, we used anti-PfHsp70 antibody (kindly provided by Dr. Nirbhay Kumar, Tulane University). We used a PfHsp70 primary antibody at 1:1,000 dilutions and anti-mouse secondary antibody (Promega) at 1:10,000 dilutions. Western blotting was performed as previously described [9 (link)]. Proteins were visualized by an enhanced chemiluminescence system (Pierce) and the band intensities were quantified by ImageJ software.