Anti rabbit secondary antibody

The right brain cortex and retina protein samples (10 μg) were subjected to 8–12% SDS-PAGE and then transferred to PVDF membranes (Merk Millipore, Burlington, MA, USA). Blocked with 5% normal goat serum (NGS) for 2 h, the membranes were incubated with primary antibodies: rabbit Anti -NLRP3 (1:1000, Cat# ab263899; Abcam), rabbit anti-GSDMD (1:500, Cat# AF-4012; Affinity), rabbit anti-caspase-1 (1:1000, Cat# AF-4022; Affinity), and rabbit anti-β-actin (1:200; Cat# AF5003; Beyotime Biotechnology, Shanghai, China) at 4 °C for 24 h. The membranes were then incubated with anti-rabbit secondary antibodies (1:500; Cat# A0239; Beyotime Biotechnology, Shanghai, China) at 4 °C for 2 h. The protein signals were detected by Immobilon Western Chemiluminescent HRP substrate (Merk Millipore, Burlington, MA, USA) and analyzed using ImageJ analysis software (https://imagej.net/Fiji/Downloads, accessed on 30 May 2017, Bethesda, MD, USA).

After fixing with 4% paraformaldehyde (Beyotime, Hangzhou, CA, China) overnight at 4 °C, the bovine ovaries were embedded with paraffin and cut into 5 µm sections for immunostaining. The ovary sections were dewaxed and dehydrated with sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH = 6) for antigen retrieval. Then, the sections were blocked for 1 h at room temperature in QuickBlock™ blocking buffer (Beyotime, Hangzhou, China) and incubated with anti-LRH-1 antibody (1:200) or non-immune rabbit IgG (1:200, for negative controls) overnight at 4 °C, respectively. Incubated antibodies were diluted with TBST (20 mM Tris-HCl, 150 mM NaCl, 0.05% Tween 20) containing 5% (w/v) non-fat dry milk. After washing three times for 5 min each time with TBST, the sections were incubated for 1 h at room temperature with anti-rabbit secondary antibody (1:5000; Beyotime, China). The samples were stained for 10 min with SignalStain^® Boost IHC Detection Reagent (Cell signaling, Danvers, MA, USA). After washing, the sections were counterstained for 20 s with haematoxylin. Finally, the stained section images were captured by a digital microscope (Nikon, Tokyo, Japan). The mean density of LRH-1 was analyzed with Image J software (National Institutes of Health, Bethesda, MD, USA).

Cells were lysed in a lysis buffer containing protease inhibitors for 30 min at 4°C. The samples were separated by 10% SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% skimmed milk in TBST solution [50 mM Tris, pH 8.0, 150 mM NaCl and 0.1% Tween-20 (v/v)] for 1 h at room temperature, and then probed by specific primary antibodies overnight at 4°C followed by anti-rabbit secondary antibody (1:5000; Beyotime) and anti‐mouse secondary antibodies (1:5000; Beyotime) for 1 hour at room temperature. The signals were detected by an enhanced chemiluminescence (ECL) reagent.
To ascertain whether PRL-3 can phosphoylate β3-tubulin, the silencing vector pGenesil-1-PRL-3 or overexpressing vector pIRES2-EGFP-PRL-3 was transfected into 293T cells. The cells were harvested at 48 h post-transfection (h p.t.) and lysed in RIPA buffer. Cell lysates were resolved using SDS-PAGE and then probed by Western blot using antibodies against PRL-3 (1:1000; Rabbit, Polyclonal, DF6747, Affinity), α-tubulin (1:2000; Rabbit, Polyclonal,112241AP, Proteintech), β3tubulin (1:1000; Rabbit, monoclonal, ab215037,abcam), p-S172-β3-tubulin (1:1000; rabbit, polyclonal, ab76286, abcam), and GAPDH (1:1000; mouse, Beyotime), respectively.

Total proteins were extracted with cell lysis solution (P0013, Beyotime). Then, 40 μg of protein was separated using SDS/PAGE (10% gel), electrotransferred on to PVDF membranes (catalogue number VVLP02500, Millipore), and incubated with rabbit anti-human Cx43 antibody (1:1000 dilution, catalogue number BA1727, Boster) or rabbit anti-human C/EBPα antibody (1:1000 dilution, catalogue number sc-61, Santa Cruz Biotechnology) at 4°C overnight. Then, anti-rabbit secondary antibody (1:2500 dilution, catalogue number P0110, Beyotime) was incubated for 1 h in room temperature. After three washes, protein bands were quantified from the membrane by densitometry using the Adobe Photoshop version 7.01 imaging system.

Total proteins were extracted with cell lysis buffer. Then, 50 μg of protein was separated using SDS/PAGE (12% gel), electrotransferred to PVDF membranes (Millipore) and incubated with rabbit anti-human Cx43 antibody (1:1000, Boster) and then anti-rabbit secondary antibody (1:2500, Beyotime). Protein bands were quantified from the membrane by densitometry using the Adobe Photoshop V7.01 imaging system.

The cells were fixed with 4% paraformaldehyde in the proportion of 1 mL of 4% paraformaldehyde for every 2 × 10⁶ cells and fixed at room temperature for 10 to 15 min. Cell lotion was added, and the mixture was centrifuged at 1,000 rpm for 5 min. After washing several times with PBS, the cells were suspended with a film breaker (permeabilization buffer 10×; ThermoFisher Scientific, Waltham, MA) and added into a 1.5-mL EP tube (1 × 10⁶/tube, 100 μL/tube). The number of cells per tube of blank control, isotype control, and sample were 1 × 10⁶/tube. We then added 5 to 20 μL isotype antibody and target antibody to each tube, respectively (refer to the instruction for specific usage); mixed thoroughly; and then incubated at 4°C for 30 min or at room temperature for 30 min in winter. The reaction tube was shaken every 10 min during incubation, so that the cells and antibodies could fully react. After several washes with film breaker, the cells were further incubated with anti-rabbit secondary antibody (1:1,000; Beyotime, Shanghai, China) labeled with fluorescent (1:200; Proteintech, Wuhan, China) for 30 min at room temperature. Finally, the cells were washed with film breaker three times and then tested with flow cytometry.