Ecl western blotting substrate

Briefly, total protein of glioma cell lines was extracted using the ExKine Total Protein Extraction Kit (Abbkine) in accordance with its protocol. Equal amounts of protein (30 μg/lane) were separated by 10% SDS‐PAGE and subsequently transferred to PVDF membranes (EMD Millipore). After blocking for nonspecific binding, the membranes were incubated with antibodies for RPN2 (1:200 dilution; ab244399; Abcam), TCF4 (1:10,000 dilution; ab76151; Abcam), c‐myc (1:1000; ab39688; Abcam), cyclin D1 (1:10,000; ab134175; Abcam) or β‐actin (1:5000; ab6276; Abcam) overnight at 4°C and followed by incubation with secondary antibodies for 1 h at room temperature. Eventually, an ECL western blotting substrate (Promega) was used to develop the protein bands.

SiHa cells were harvested at 24 h post-transfection and cells were counted. Cell pellets containing 10⁵ SiHa cells were resuspended in 1 mL RIPA solution (Cell Signaling Technology) to extract total proteins. Concentrations of total proteins were measured using BCA assay (Cell Signaling Technology). In a 95°C incubator, protein samples were denatured for 10 min, followed by electrophoresis using 12% SDS-PAGE gel. After that, proteins were transfected to PVDF membranes and blocking was performed in 5% non-fat milk (PBS) at room temperature for 1 h. GAPDH (1:1800, ab37168, Abcam) and CDK6 (1:1800, ab151247, Abcam) rabbit polyclonal primary antibodies were used to incubate with the membranes at 4°C for 18 h, followed by incubation with HRP (IgG) (1:1800; ab6721; Abcam) goat anti-rabbit secondary antibody at 24°C for 2 h. ECL Western Blotting Substrate (Promega Corporation) was dropped onto the membranes and MYECL™ Imager (Bio-Rad) was used to detect signals. Image J v1.46 software was used for data normalizations.

Equal amount of proteins (50 μg) were separated onto SDS‐polyacrylamide gels and were electrotransferred to PVDF membranes (Millipore, Bedford, MA, USA). The membranes were immunoblotted overnight at 4°C with primary antibodies, followed by their respective secondary Abs; GAPDH was used as the loading control. Primary Abs used included anti‐FOXC1 (1:500, AP8907B; Abgent, San Diego, CA, USA), anti‐CXCR4 (1:1000, ab58176; Abcam, Cambridge, UK), anti‐PCMT1 (1:1000, ab97446, Abcam), anti‐GAPDH (1:2000, sc‐47724; Santa Cruz Biotechnology, Dallas, TX, USA). After that, the membranes were incubated with corresponding HRP‐conjugated secondary Abs. The blot signals were visualized using the ECL western blotting substrate (Promega).

Six days after transduction native rMSCs, rMSCs-Katushka, rMSCs-HEXA-HEXB, native hMSCs, hMSCs-Katushka, hMSCs-HEXA-HEXB were trypsinized and washed three times with saline. Next, a western blot analysis was conducted, by the protocol described in our previous research (Shaimardanova et al., 2022). In brief, RIPA buffer containing proteinase and phosphatase inhibitors was used for protein extraction. Protein concentration in the sample was determined using the Pierce^TM BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA, Cat# 23225). Primary rabbit polyclonal antibodies to HEXA (dilution 1:300, Cloud-Clone Corp., Houston, TX, USA, Cat# PAA195Hu01, RRID: AB_2936290) and HEXB (dilution 1:250, Cloud-Clone Corp., Cat# PAA637Hu01, RRID: AB_2936291) and secondary goat polyclonal antibodies to rabbit immunoglobulin G conjugated with horseradish peroxidase (dilution 1:1000, Sigma-Aldrich, Cat# A6154, RRID: AB_258284) were used for the analysis. The membranes were incubated with primary antibodies for 12 hours at 4°C, with secondary antibodies for 2 hours at room temperature. For detection and visualization, ECL Western Blotting Substrate (Promega, Madison, WI, USA, Cat# W1001) and ChemiDocXRS⁺ instrument (BioRad, Hercules, CA, USA) were used. The experiment was carried out in three biological and three technical replicates 24 hours after transduction.