Horseradish peroxidase conjugated anti rabbit igg secondary antibody

Purified genomic DNA was denatured in 0.4 M NaOH, 10 mM EDTA at 95 °C for 10 min, then neutralized with ice-cold 2 M ammonium acetate (pH 7.0). Two-fold serial dilutions of the denatured DNA samples were generated and spotted on a nitrocellulose membrane by using an assembled Bio-Dot apparatus (Bio-Rad) according to the manufacturer’s instructions. A synthetic oligonucleotide with a known amount of 5hmC was used as standard^{1 (link)}. The membrane was washed with 2xSSC buffer briefly, air-dried and vacuum-baked at 80 °C for 2 h. DNA hybridized membrane was blocked with 5% non-fat milk for 1 h at room temperature and incubated with an anti-5hmC antibody (1:3000, Active Motif) overnight at 4 °C. Next day, the membrane was incubated with a horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody (1:10,000; Sigma) for 1 h at room temperature. The membrane was visualized by West-Q Pico Dura ECL Solution (GenDEPOT). The membrane was washed with 1× TBST briefly and then stained with 0.02% methylene blue in 0.3 M sodium acetate (pH 5.2) to confirm the total amounts of loaded DNA samples. The uncropped and unprocessed scans of the blots are available in source data file.

HEK293T cells were grown in 12-well plates. After transfection, they were lysed, and proteins were extracted in ice-cold RIPA buffer (50 mM Tris [pH 7.5], 150 mM NaCl, 1% NP-40, 0.5% NaDOC, 0.1% sodium dodecyl sulfate [SDS], and 1 mM EDTA) that contained mini complete protease inhibitor cocktail (Roche) and phosphatase inhibitors (Sigma). Lysates were centrifuged at 12,000 × g for 10 min. Protein extracts (20 μg) were separated by 10% or 8% SDS-PAGE, transferred to a Protran nitrocellulose membrane (Whatman), and blocked for 2 h at room temperature in TBS-T (50 mM Tris–HCl [pH 7.5], 150 mM NaCl, and 0.1% Tween 20 plus 5% dry nonfat milk). The nitrocellulose sheets were then incubated at 4 °C overnight in blocking solution with a monoclonal antibody against SIP (1:1000; catalog no. ab51288, Abcam) or polyclonal antibody against N-terminal HTT (1:6000; catalog no. H7540, Sigma). As a control, a polyclonal antibody against GAPDH (1:1000; catalog no. sc-25778, Santa Cruz Biotechnology) or monoclonal antibody against p-cadherin (1:2500; catalog no. ab6528, Abcam) was used, followed by incubation with horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody (1:10,000; catalog no. A0545, Sigma) for 3 h at room temperature. The signal was detected using an enhanced chemiluminescence substrate (Amersham Biosciences).

Proteins extracted from patients’ tumor tissue samples were separated by SDS-PAGE and then transferred to PVDF membrane. After incubation with primary antibodies at 4°C overnight, the membranes were washed three times with Tris-buffered saline containing Tween-20 (TBST), and then incubated with the horseradish peroxidase-conjugated secondary antibody (anti-rabbit IgG: 1:4000, Sigma, USA) for 2 h at room temperature. The membranes were washed again in TBST and visualized using an Enhanced Chemiluminescence Kit (PerkinElmer). The rabbit anti-HADH (1/1000) was from Thermo Fisher Scientific. The anti-p-Akt, Akt (Ser-473, 1/1000), and GAPDH antibodies (1/8000) were from Cell Signaling. The band density was quantified by ImageJ and normalized to GAPDH.

Kidney samples were run on polyacrylamide minigels (Burnette 1981 (link)). After transfer by electroelution to nitrocellulose membranes (GE Healthcare Limited, Little Chalfont, UK), blots were blocked with 5% nonfat dry milk in Tris-buffered saline solution. Blots were then incubated overnight with antibodies against AQP2 (1:2000; Santa Cruz Biotechnology, Santa Cruz, CA); actin (1:5000; Santa Cruz Biotechnology); p21 (1:500; Santa Cruz Biotechnology); VDR (1:500; Santa Cruz Biotechnology). The labeling was visualized with horseradish peroxidase-conjugated secondary antibody (anti-rabbit IgG, 1:2000, or anti-goat, 1:10000; Sigma Chemical, St. Louis, MO) and enhanced chemiluminescence (ECL) detection (Amersham Pharmacia Biotech, Piscataway, NJ).

Tissue and cell proteins were extracted by RIPA lysis buffer and separated by SDS-PAGE. Subsequently, the protein was transferred to PVDF membrane and incubated with a primary antibody (GAPDH, NLRP3, Cleaved caspase-1, Pro caspase-1, Cleaved IL-1β, p-IκBα, IκBα, p-NF-κB p65, NF-κB p65 (1 : 1000, CST, Boston, USA) and ASC1 (1 : 1000, Abcam, Cambridge, USA)) overnight at 4°C. The membrane was then incubated with horseradish peroxidase-conjugated secondary antibody (anti-rabbit IgG, 1 : 10000, Sigma, St. Louis, USA) for 1 h. Finally, the protein bands were detected by an ECL kit and quantized using Quantity One 1-D Analysis Software (Bio-Rad, Hercules, USA).

Samples of membrane fractions were run on 12.5 % polyacrylamide minigels (for AQP5), 10 % polyacrylamide minigels (for α-ENaC, α1-Na,K-ATPase, and TLR4), or 8 % polyacrylamide minigels (for NKCC1). After transfer by electroelution to nitrocellulose membranes (PolyScreen, PVDF Transfer; Life Science Products, Boston, MA, USA), blots were blocked with 5 % milk and 0.1 % Tween 20 in phosphate-buffered saline for 1 h, then incubated with anti-AQP5 antibody (1:500), NKCC1 antibody (1:1000), α₁-Na,K-ATPase antibody (1:500), α-ENaC antibody (1:100), or TLR4 antibody (1:100). The labeling was visualized with horseradish peroxidase-conjugated secondary antibody (anti-rabbit IgG, diluted 1:5000, or anti-goat IgG, diluted 1:10,000; Sigma) using the enhanced chemiluminescence detection system (Amersham Pharmacia Biotech, Piscataway, NJ, USA). Bands corresponding to protein expression of AQP5, α-ENaC, α1-Na,K-ATPase, NKCC1, TLR4, and actin were quantified by densitometric analysis using Image J software (Research Services Branch, National Institutes of Health, Bethesda, MD, USA). Bands were normalized to actin and are expressed as percentages of the control values.

Proteins were separated on SDS-polyacrylamide minigels by electrophoresis [19] (link). After transfer by electroelution to polyvinylidene difluoride (PVDF) membranes (GE Healthcare Limited, Little Chalfont, UK), blots were blocked for 60 minutes with 5% non-fat dry milk in Tris-buffered saline solution. Blots were then incubated overnight with antibodies against Actin (1∶5,000), VDR (1∶500), Klotho (1∶500) and TGF-beta (1∶200) (Santa Cruz Biotechnology, CA, USA). The labeling was visualized with horseradish peroxidase-conjugated secondary antibody (anti-rabbit IgG, diluted 1∶2,000, or anti-goat, diluted 1∶10,000, Sigma Chemical, St. Louis, MO, USA) and enhanced chemiluminescence (ECL) detection system (GE Healthcare Limited, Little Chalfont, UK).