Ab181150

WB was performed according to the previously reported methods, with some modifications [21 (link)]. Cells were harvested in RIPA lysis buffer (P0013B, Beyotime, Shanghai, China) that was supplemented with complete protease inhibitors (Sigma, Darmstadt, Germany). Cell lysates were separated on 10% SDS-PAGE and blotted onto the NC membrane. The membrane was then blocked in 5% nonfat milk and incubated with the primary antibodies overnight at 4 °C in the following dilutions: anti-G3bp1 (1:3000, ab181150, Abcam, Cambridge, UK), anti-G3bp1 (1:3000, ab56574, Abcam, Cambridge, UK), anti-G3bp2 (1:3000, NBP1-82977, Novus biologicals, Abingdon, UK), anti-Ubiquitin (1:1000, sc-8017, Santa Cruz, Dallas, TX, USA), anti-HSP70 (1:10000, ab45133, Abcam, Cambridge, UK), anti-VCP (1:3000, ab109240, Abcam, Cambridge, UK), anti-LC3 (1:1000, A19665, ABClonal, Wuhan, China), and anti-GAPDH (1:10000, KC-5G5, Kangchen Biotech, Shanghai, China). After the membranes were washed with 0.1% PBST, HRP-conjugated anti-mouse (7076S, CST, Danvers, MA, USA) or anti-rabbit (7074S, CST, Danvers, MA, USA) antibody was used as the secondary antibody for 1 h at room temperature. The signals were detected by using the Western ECL Substrate (170-5060, Bio-Rad, Irvine, CA, USA). The images were visualized by using the Bio-Rad ChemiDoc XRS+ system.

All tissues are examined pathologically by two pathologists. Serialized sects (4 microns) is cut from paraffin-embedded GC tissues or mice tumour tissues. All sections are dewaxed, hydrated, endogenous enzyme removal, and antigen retrieval. Subsequently, Anti-GPC3 antibody(1:500; Abcam, ab216606, ab181150), Anti-αSMA antibody (1:200; Abcam, ab124964, ab240654), and related fluorescent secondary antibody (ThermoFisher Scientific). To determine the expression of GPC3, we calculated the mean integral optical density (IOD) per slice using Image-Pro Plus 6.0 software (Media Cybernetics, USA).

For immunostaining, cells grown on PDL-coated glass coverslips were fixed with 4% paraformaldehyde for 15 min and permeabilized (0.2% Triton X-100 and 50 mM NH₄Cl in PBS) for 5 min. After blocking (30 min, 2% fetal bovine serum, 2% serum albumin, and 0.2% fish gelatin in PBS), the coverslips were incubated in primary antibody solution at RT for 1 h and washed with PBS. Finally, the cells were incubated in Alexa-coupled secondary antibody solution and treated with DAPI or TO-PRO-3 for staining of the nuclei. Antibodies and reagents used were anti-RPS6 (sc-74459; Santa Cruz Biotechnology), anti-G3BP1 (ab181150; Abcam), anti-fibrillarin (ab5821; Abcam), DAPI (Roche Applied Science), and TO-PRO-3 (Thermo Fisher Scientific). Single-plane images were obtained on a confocal laser scanning LSM710 microscope (Carl Zeiss) with a 63× or 40× immersion objective. Image editing and particle analysis was carried out using ImageJ software, and for statistical analysis, GraphPad Prism (version 7.01) software was used.

Visceral adipose tissues were prepared and lysed according to standard protocols. Antibodies to G3BP (ab181150, 1:1000) and GAPDH (ab8245, 1:2000) were purchased from Abcam. Blotting membranes were incubated with the primary antibody at 4°C overnight and the secondary anti-rabbit IgG (#32731; 1:10000; Thermo Scientific) at room temperature for 1 h. The resulting bands were visualized using a Tanon 5500 imaging system (Tanon, Shanghai, China). The results were quantified using ImageJ software (National Institute of Mental Health, USA).

Cells were lysed in lysis buffer (P0013, Beyotime) with a protease inhibitor cocktail (100 ×) (5871, Cell Singling Technology) for 20 min on ice, followed by centrifugation at 12000 × g for 20 min at 4 °C. Proteins were separated by SDS–PAGE and transferred to a PVDF membrane (RINB77899, Millipore). The membrane was blocked for 1 h in TBS plus 5% nonfat milk, followed by incubation at 4°C overnight or at room temperature (RT) for 2–3 h with primary antibodies. After three washes with TBST, the membrane was incubated with secondary antibodies at RT for 1 to 2 h. Images were obtained using Odyssey software (Gene). Antibodies used in this study include: anti‐G3BP1 (1:1000; ab181150, Abcam), rabbit anti‐G3BP2 (1:1000; HPA018425, Sigma), rabbit anti‐AKAP12 (1:1000; ER1903‐55, HUABIO), GAPDH (1:1000; HC301‐01, TransGen Biotech); rabbit anti‐HDAC6 (1:1000; 6712S, Cell Signaling Technology). Secondary antibody: conjugated with 800 nm fluorophores (SeraCare KPL, 1:10000).

Before immunofluorescence assay, cells were added with prehybridization solution at 37 °C for 1 h. After removing the prehybridization solution, the hybridization solution containing the probe of SNORA71 or ROCK2 was added and hybridized overnight. After washing with SSC, cells were incubated with primary antibody anti‐G3BP1 (1 : 500; # ab181150; Abcam) and secondary antibody: primary antibody were incubated overnight at 4 °C, then washed with PBS for 3 × 5 min; Corresponding secondary antibody was dropped and incubated at room temperature for 50 min, then washed with PBS for 3 × 5 min. DAPI dye for dyeing nuclear for 8min away from light. Images were obtained using a fluorescence microscope.

Cultured cells were allotted a 24-hour growth period atop cover glass substrates. The initiation of the FISH assay unfolded with the application of a JAK2 mRNA FISH probe mix kit, thoughtfully crafted by RiboBio Technology (Guangzhou, China). Following the FISH experiment, the sequential undertaking of immunofluorescence ensued. A rabbit monoclonal antibody directed against G3BP1 (catalog: ab181150, Abcam, The United Kingdom) was entrusted with an overnight incubation period. Subsequent to this, the cells bore witness to staining with Horseradish Peroxidase goat anti-rabbit IgG-R (Dylight 488, Goat Anti-Rabbit IgG, catalog: #A23220, Abbkine). The cell nucleus was artfully stained with DAPI, imparting visual clarity to the ensuing images.