Glut1 primary antibody

PCa cells seeded in 100-mm cell culture dishes were treated with Etn for different time points. After treatment, cells were subjected to immunoblotting as described previously 33 (link). Lysates from fresh tumor samples were prepared using BeadBlaster microtube homogenizer (LabRepCo) following the manufacturer's guidelines. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then were transferred onto polyvinylidene difluoride (PVDF) membranes. Membranes were incubated with anti-light chain 3 primary antibody (LC3; Cell Signaling Technologies, #2775S, 1:500) or GLUT1 primary antibody (Abcam, #ab115730, 1:1000) overnight at 4 °C; anti-beta-actin primary antibody (Santa Cruz Biotechnology, #SC-47778, 1:1000) was used as a loading control. Subsequently, membranes were incubated with goat anti-mouse (Santa Cruz Biotechnology, #sc-516102, 1;8000) or goat anti-rabbit (Santa Cruz Biotechnology, #sc-2357, 1:8000) IgG horseradish peroxidase-conjugated secondary antibodies. All antibodies against pro-apoptotic proteins were from Pro-Apoptosis Bcl-2 Family Antibody Sampler Kit (Cell Signaling Technologies, #9942T). The signal was visualized using the Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, #32106). Signal intensities were quantified using ImageJ and were normalized to the respective β-actin signal intensity.

SW948 and SW1116 cells were treated with 3 mM of metformin for 48 h at a seeding density of 1.0 × 10⁶ cells per well using HG and LG media. After treatment, cells were trypsinized and washed twice with PBS before adding 4% Paraformaldehyde (PFA) fixation and incubating on ice. The fixed cells were subsequently incubated with GLUT1 primary antibody (Abcam, Cambridge, United Kingdom) at the concentration 1:500 for 1 h at room temperature. GLUT1 labelled cells were washed twice in PBS and labelled with Alexa fluor 647 conjugated Donkey anti-rabbit secondary antibody (Abcam, Cambridge, United Kingdom) for another 30 min before analysing with Accuri C6 flow cytometer (BD Biosciences, San Jose, USA).