Ab9332

For the in vitro kinase assays, 2 μg of dephosphorylated BCAP (FL) or 100 μg of dephosphorylated myelin basic protein (31314; Active Motif) were diluted in 500 μl of kinase buffer (50 mm HEPES, 10 mm MgCl₂, 0.01% BRIJ35, 1 mm EGTA, and 150 μm ATP, pH 7.5). Upon adding 60 pmol of SYK (PV3857; Thermo Fisher Scientific), LYN (PV6448; Thermo Fisher Scientific), BTK (PV3363; Thermo Fisher Scientific), TYK2 (PV4790; Thermo Fisher Scientific), ITK (PV4193; Thermo Fisher Scientific), CSNK1A1 (PV3850; Thermo Fisher Scientific), or CSNK2A1 (PV3248; Thermo Fisher Scientific), the samples were incubated at 30 °C for 30 min. The reaction was stopped using 4× SDS loading dye, and the samples were analyzed using Western blotting. For chemiluminescence detection, anti-BCAP (AF4857; R&D Systems), anti-phosphotyrosine (Ab179530; Abcam), anti-phosphoserine (Ab9332, Abcam), anti-rabbit IgG-HRP (A0545; Sigma), and anti-goat IgG-HRP (A5720; Sigma) were used.

Proteins were extracted from tissues and cells with cell lysis buffer, boiled for 5 min, and stored at -80°C until use. Samples were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrically transferred to polyvinylidene fluoride (PVDF) membranes blocked with 5% nonfat dry milk in TBS-Tween (blocking buffer) for 1 h. PVDF membranes were probed at 4°C overnight in blocking buffer with the following primary antibodies: MSTN (ab201954, Abcam, USA), p-Smad2 (ab280888, Abcam, USA), p-Smad3 (ab52903, Abcam, USA), Smad2 + Smad3 (ab202445, Abcam, USA), GLUT1 (A11727, ABclone, China), GLUT4 (A7637, ABclone, China), p-AMPKα1+α2 (ab133448, Abcam, USA), G6PD (ab993, Abcam, USA), TKL (1 : 1000, ab181235, Abcam, USA), RPI (ab137629, Abcam, USA), phosphoserine (ab9332, Abcam, USA), phosphotyrosine (ab10321, Abcam, US), p-AKT (ab38449, Abcam, USA), p-P38 (ab31828, Abcam, USA), and α-tubulin (11224-AP, Proteinch, China). Membranes were washed three times and incubated with secondary antibody diluted 1 : 5000 in blocking buffer. Finally, protein expression was detected and recorded.

The procedures of western blot assay have been described previously.²⁷ Antibodies that recognize AMD1 (11052‐1‐AP), Tubulin (11224‐1‐AP), NANOG (14295‐1‐AP), SOX2 (11064‐1‐AP), KLF4 (11880‐1‐AP), OCT4 (11263‐1‐AP), METTL3 (15073‐1‐AP), METTL14 (26158‐1‐AP), FTO (27226‐1‐AP), and ALKBH5 (16837‐1‐AP) were purchased from Proteintech (Wuhan, China). Antibodies that recognize IQGAP1 (ab86064), phosphoserine (ab9332), phosphothreonine (ab9338), and SPM (ab7318) were obtained from Abcam (Cambridge, MA, USA). Secondary antibodies were obtained from Beyotime Biotechnology (Shanghai, China).

Protein fractionation in the chromatin-bound, the nucleoplasmic or the cytoplasmic compartments was performed essentially as described [81 (link)]. The protein extraction was performed on 4 × 10⁷ primary MEFs (passage 3) or on 4 × 10⁷ R1 mouse ESCs. The protein fractions were separated on 4–20 % gradient Bis-Tris SDS gels (BioRad), blotted, and incubated with the following primary antibodies: HP1β (1MOD-1A9, Euromedex; 1:2000), HP1γ (2MOD-1G6, Euromedex; 1:2000), H3K9me3 (rabbit polyclonal; 1:100), kindly provided by T. Jenuwein (Freiburg), histone H3, kindly provided by M. Bustin (1:10,000, rabbit), and alpha tubulin (#ACLX135B, Accurate Chemical & Scientific Corporation). Other antibodies used for western blots included lamin A/C (sc-20680, SantaCruz; 1:100), hnRNPa2/b1 (ab31645, Abcam; 1:200), phosphoserine (ab9332, Abcam; 1:100) and phosphothreonine (Cell Signaling #93865; 1:3000).

Brain hippocampus tissues were centrifuged at 12,000 g for 10 min following homogenization in RIPA lysis buffer (1:10, w/v). The super-natants were collected. The whole protein samples were obtained by this method. The nucleus and cytoplasm protein were obtained by using the nucleus and cytoplasmic extraction kit. (Boster Biological Technology Co.,Ltd) All protein samples were determined by BCA protein assay kit. Make appropriate dilution of serum and tissue samples based on the protein concentration. Then the samples were mixed with 5× loading buffer, then were boiled at 100◦C for 5 min and stored at − 70◦C until further analysis.
Proteins were run on 10% or 12% polyacrylamide gels and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% BSA for 60–90 min at room temperature and incubated with antigen-specific primary antibodies overnight at 4 °C. Blots were then incubated with species-specific secondary antibodies for 60 min at room temperature. Proteins were visualized by incubation with a chemiluminescent substrate kit. The primary antibodies used in this study were anti-HMGB1 (10829-1-AP, Proteintech, USA), anti-p-serine (ab9332, Abcam, UK), anti-NF-κB (10745-1-AP, Proteintech, USA), anti-p-38 (14064-1-AP, Proteintech, USA), anti-PP2A (13482-1-AP, Proteintech, USA), and anti-PKC (21991-1-AP, Proteintech, USA).