Ab205718

Proteins from samples were isolated using RIPA buffer (Vazyme, Nanjing, China) and were segregated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and then proteins were transferred onto the polyvinylidene difluoride membranes (Vazyme). The membranes were blocked with 5% skimmed milk (Vazyme). Thereafter, the membranes were incubated with the primary antibodies: anti-Irak2 (1:2000, ab62419, Abcam, Cambridge, UK) or glyceraldehyde 3-phosphate dehydrogenase (1:2500, ab9485, Abcam) overnight. After being rewashed, the membranes were incubated with the secondary antibody (1:3000, ab205718, Abcam) for 2 h. The membranes were analyzed by the ChemiDoc™ MP Imaging System (Bio-Rad) after being treated with ECL kit (Vazyme).

Protein levels were detected by western blotting referring to the protocols in a previous report [20 (link)]. In brief, HaCaT cells were lysed in radio-immunoprecipitation assay buffer (Beyotime, Shanghai, China), and extracted protein was quantified by bicinchoninic acid assay kit (Thermo Fisher Scientific). Protein samples (20 μg) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transfer of polyvinylidene fluoride membranes (Thermo Fisher Scientific). After incubating in 5% fat-free milk, the membranes were incubated with primary antibodies for IGF2BP2 (ab129071, 1:3000 dilution, Abcam, Cambridge, UK), HPSE (ab254254, 1:3000 dilution, Abcam), or β-actin (ab8227, 1:3000 dilution, Abcam) overnight, and then incubated with horseradish peroxidase-conjugated IgG (ab205718, 1:8000 dilution, Abcam) for 2 h, followed by exposure to enhanced chemiluminescence kit (Beyotime). The visualized blots were analyzed via Image J software (NIH, Bethesda, MD, USA), with β-actin as a reference.

50 nM APC/C, 50 nM Cdc20, 200 nM Cyclin B1, 30 nM UBA1, 20 µM Ubiquitin, 5 mM ATP, 0.25 mg/mL BSA and 250 nM UbcH10 were mixed in a 15 µL reaction volume with reaction buffer: 40 mM HEPES pH 8.0, 0.6 mM DTT, 10 mM MgCl₂. 270 nM of each MCC construct was added into its respective reaction. The reaction mixture was incubated at 25 °C for 15 minutes. Samples were taken at 0 and 15 min, and the reaction was terminated with SDS/PAGE loading dye. The reaction mixture was run on a 4–12% NuPAGE Bis-Tris gel and transferred to a nitrocellulose membrane for Western blotting. Blocking was carried out in 5% BSA-TBST and washed in 1× TBST. Ubiquitin-modified Cyclin B1 was detected using an anti-CyclinB1 mAb (ABclonal, A19037) at a 1:2,000 dilution, and HRP-conjugated goat anti-rabbit secondary antibody (Abcam, ab205718) at a 1:10,000 dilution. The membrane was incubated with ECL, detected using the ImageQuant 800 (Amersham), and quantified using ImageJ.

Proteins were extracted with RIPA buffer, uploaded on 10% SDS-PAGE gel, and transferred to PVDF membrane. Membrane was blocked with 5% non-fat milk for 2 h, and then incubated with antibodies (Abcam, Cambridge, CA, USA), including anti-SLC1A5 (1:1,000, ab237704), anti-Bcl-2 (1:1,000, ab32124), anti-Bax (1:1,000, ab32503), anti-YY1 (1:1,000, ab109228), anti-Nanog (1:200, ab21624), anti-Sox2 (1:1,000, ab97959), anti-GAPDH (1:2,500, ab9485), and secondary antibody (1:50,000, ab205718). ECL reagent (Beyotime) was then used for visualizing protein bands, and gray value was analyzed by Image J software.

Flow cytometry was used to detect the expression of WASP in peripheral blood monocytes. Using human lymphocyte separation medium (Solarbio, Beijing, China) density gradient centrifugation, PBMCs were isolated from the peripheral blood of the patient and his parents. Using IntraPrep Permeabilization Reagent (Beckman Coulter, A07803), cells were fixed for 15 minutes and permeabilized for 15 minutes, respectively. Next, add the 1 : 100 dilution of rabbit anti-WASP (Abcam, ab75830, UK) to the samples and incubate for 30 min at room temperature. Finally, add 1 : 2000 diluted goat anti-rabbit IgG H&L (fluorescein isothiocyanate; Abcam, ab205718) and incubate for 25 min in the dark. After the cells were centrifuged and resuspended, we used the NAVIOS Flow Cytometer (Beckman Coulter) to detect the expression of WASP, and the data were analyzed by NAVIOS software.

Cardiac GPs, atria and ventricles were fixed with 10% formalin and embedded in paraffin. Then, 5-μm-thick tissues were serially cut from paraffin blocks and mounted onto glass slides coated with 1% (W/V) gelatin solution. After antigen retrieval, the tissue sections were incubated with primary antibodies against Na_V1.8 α subunit (1:20, ab114110, Abcam, Cambridge, UK) at 4°C overnight. After washing three times with PBS, the sections were then incubated with the secondary antibody (HRP labeled goat anti rabbit, 1:200, ab205718, Abcam, Cambridge, UK) expression were detected using DAB (brown) staining.

Proteins in H9c2 cells were extracted by lysis buffer (P0013J, Beyotime). After determining the concentration with a BCA detection kit (P0012, Beyotime), the proteins were loaded and electrophoresed on 10% SDS polyacrylamide gel (P0012AC, Beyotime), and then transferred onto PVDF membranes (FFP26, Beyotime,). After using 5% non-fat milk to block membranes for 60 min at 37°C, the following primary antibodies were incubated with membranes overnight at 4°C: Bcl-2 (ab59348, 1:1000, 26 kDa, Abcam, USA), Bax (ab32503, 1:1000, 21 kDa, Abcam), Cleaved caspase-3 (ab49822, 1:500, 17 kDa, Abcam), iNOS (ab3523, 1:200, 135 kDa, Abcam), Cox-2 (ab15191, 1:250, 69 kDa, Abcam), CDK4 (ab199728, 1:2000, 34 kDa, Abcam), Cyclin D1 (ab16663, 1:25, 36 kDa, Abcam), HSP70 (ab181606, 1:1000, 70 kDa, Abcam), and GAPDH (ab181602, 1:10000, 36 kDa, Abcam). Following extensive washing, protein bands were incubated with the secondary antibody: Goat Anti-Rabbit IgG H&L (HRP) (ab205718, 1:2000, 42 kDa, Abcam) at 37°C for 2 h. The detection of signal was performed according to a standard ECL method (27), and analysis software (Image J 1.5i, National Institutes of Health, USA) was used for images to measured protein expression. GAPDH was used as housekeeping gene.