Wlp2412

Western blotting was performed as previously described [26 (link)]. In brief, a total cell protein extraction kit (KeyGen Biotechnology, Nanjing, China) was used to isolate the total proteins of glioma tissues or GSCs. Then, protein lysates were prepared, and the total protein for each sample was transferred onto the polyvinylidene difluoride (PVDF) membranes after SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and blocked 2 h at room temperature with 2% bovine serum albumin (KeyGen Biotechnology). Subsequently, all these membranes were incubated overnight at 4 °C with the primary antibodies as below: HOXB5 (1:1000; #ab109375, Abcam), IL6 (1:1000; #ab233551, Abcam), p-JAK2 (1:500; #WL02997, Wanleibio, Shenyang, China), JAK2 (1:500; #ab195055, Abcam), p-STAT3 (1:1000; #WLP2412, Wanleibio), STAT3 (1:2000; #ab76315, Abcam), SRSF1 (1:500; #12929-2-AP, Proteintech, Rosemont, IL, USA) and β-actin (1:2000; #20536-1-AP, Proteintech). Following 2 h’ secondary antibodies (1:1000; #SA00001-2, Proteintech) incubation, all the bands were detected by a chemiluminescence ECL kit (Beyotime Biotechnology, Beijing, China) and quantified by Image J software (National Institutes of Health, Bethesda, MD, USA). The relative expression was calculated based on the internal control β-actin.

HUVECs were homogenized in RIPA lysis buffer and centrifuged at 20,000
g for 20 min at 4°C. Protein samples (10 μg) from independent experiments were electrophoresed on a 7.5%‒15% SDS-polyacrylamide gel and transferred onto PVDF membranes. Blots were blocked with 5% non-fat milk for 1 h at 37°C and incubated overnight at 4°C with antibodies against cystathionine gamma-lyase (CSE; sc-374249; Santa Cruz, Santa Cruz, USA), 3-mercaptopyruvate sulfotransferase (MPST; sc-374326; Santa Cruz), P21 (HA500005; HuaAn, Hangzhou, China), PPARδ (A5656; ABclonal, Shanghai, China), SGLT1 (A11976; ABclonal), SGLT2 (A20271; ABclonal), cystathionine beta-synthase (CBS; 14787-1-AP; Proteintech, Rosemont, USA), P53 (80077-1-RR; Proteintech), Caspase1 (WL03450; Wanlei, Shenyang, China), eNOS (WL01789; Wanlei), GLUT1 (WL01163; Wanlei), IL1β (WL00891; Wanlei), NLRP3 (WL02635; Wanlei), STAT3 (WL01836; Wanlei), and phosphorylated STAT3 (WLP2412; Wanlei). After being washed three times with TBST, the blots were incubated with anti-rabbit (SA00001-2; Proteintech) or anti-mouse IgG-HRP (SA00001-1; Proteintech). Antigen-antibody reactions were detected using ECL reagents (MERCK, Darmstadt, Germany) and detected with the ChemiScope 6100 system (CLiNX, Shanghai, China) for densitometric analysis.

Western blotting was carried out as previously described [22 (link)]. Briefly, total protein from tissues and cells was extracted by using a total cell protein extraction kit (KeyGen Biotechnology, Nanjing, China). Then, equivalent amounts of protein were separated by SDS-PAGE and transferred onto the polyvinylidene difluoride (PVDF) and blocked with 2% bovine serum albumin (Beyotime Biotechnology, Beijing, China) for 2 h. The membrane were incubated overnight at 4 °C with the primary antibodies: β-actin (1:2000; #20536-1-AP, Proteintech), XPR1 (1:1000; #14174-1-AP, Proteintech), IL6 (1:1000; #ab233551, Abcam), IL6 (1:1000; #ab233551, Abcan), p-JAK2 (1:500; #WL02997, Wanleibio, Shenyang, China), JAK2 (1:500; #ab195055, Abcam), p-STAT3 (1:1000; #WLP2412, Wanleibio), STAT3 (1:2000; #ab76315, Abcam), IGF2BP3 (1:1000; #14642-1-AP, Proteintech), followed washing with PBS/T (Phosphate Buffer Solution with 0.05% Tween20). Next, the membranes were incubated with horseradish peroxidase-linked secondary antibody (1:1000; #SA00001-2, Proteintech) at room temperature for 1 h. Finally, immunoreactive bands were displayed by a chemiluminescence ECL kit (Beyotime Biotechnology, Beijing, China) and quantified by Image J software (National Institutes of Health, Bethesda, MD, USA) while the β-actin was used as internal control.