Ripa radioimmunoprecipitation assay buffer

H1975 cells were plated in a 6-well plate at 80% confluency in duplicates and were treated with AICAR (1 mM) for 15 min. Cells were detached using a cell scraper (CellPro) and pelleted. The supernatant was removed, and the pellets were heated for three mins at their respective temperature (37–55 °C) in a mini dry bath incubator (Four E’s Scientific), followed by a three-min cool-down. Cell pellets were then resuspended in 80 μl of RIPA (radioimmunoprecipitation assay) buffer (Thermo Fisher Scientific) supplemented with protease and phosphatase inhibitor cocktail (Roche) to lyse the cells. The mixtures were shaken at 4 °C for 2 h, followed by centrifugation at 4 °C for 40 min at 14,000 RPM. The supernatants were collected and quantified, followed by western blotting.

CDDP was purchased from Selleckchem Inc. (Houston, TX, USA). ¹³C₆-L-lysine, L-lysine, ¹³C₆¹⁵N₄-L-arginine, L-arginine, Dulbecco’s modified Eagle’s medium (DMEM)/F12 for SILAC, APE1 siRNA, dimethyl sulfoxide (DMSO), 2-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), bovine serum albumin, and Dulbecco’s phosphate-buffered saline (PBS) were obtained from Sigma-Aldrich (St. Louis, MO, USA). 6-Diamidino-2-phenylindole (DAPI), Opti-minimal Essential Medium (MEM), Lipofectamine 2000, and the negative control siRNA were purchased from Invitrogen Inc. (Carlsbad, CA, USA). The Annexin V-phycoerythrin (PE) apoptosis detection kit was purchased from BD Biosciences Inc. (San Jose, CA, USA). The Cyto-ID® Autophagy detection kit was obtained from Enzo Life Sciences Inc. (Farmingdale, NY, USA). The Western blotting substrate, Pierce™ bicinchoninic acid (BCA) protein assay kit, skim milk, and radioimmunoprecipitation assay buffer (RIPA) were purchased from Thermo Fisher Scientific Inc. (Hudson, NH, USA). The polyvinylidene difluoride (PVDF) membrane was obtained from Bio-Rad Inc. (Hercules, CA, USA). The antibody against human β-actin was obtained from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). The remaining primary antibodies for signalling proteins related to apoptosis and autophagy were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA).

Following activation and subsequent treatment, astrocyte CM was collected, transferred to 15 ml conical tubes on ice, and centrifuged at 4°C for 10 min at 16,000 rpm. CM was subsequently filtered using a 0.44 μm syringe filter unit to remove any residual cells and protein quantification using the BCA assay was performed prior to further experimental assays.
Following activation and subsequent treatment, astrocytes were washed three times in ice-cold 1× PBS and gently collected using a cell scraper. The cell suspension was transferred to Eppendorf tubes and centrifuged in a 4°C microcentrifuge at 16,000 rpm for 10 min. Media-containing supernatant was carefully discarded and the cells were lysed using ice-cold radio-immunoprecipitation assay buffer (RIPA; Thermo Scientific) supplemented with protease inhibitors according to the manufacturer’s protocol. Samples were passed through a 25 gauge needle five times until sufficiently homogenized and then centrifuged at 14,000 rpm for 15 min. The supernatant from each sample was carefully removed ensuring that the underlying pellet was not disturbed, and then transferred to a new low-bind Eppendorf tube. Protein quantification using the BCA assay was also performed.

In brief, cells were gathered in Radioimmunoprecipitation assay buffer (RIPA) (Thermo Scientific) supplemented with phosphatase–proteinase inhibitor (Thermo Scientific) and kept at 4 °C overnight. Eight micrograms of sample were run on NuPAGE 4–12% Bis-Tris gels (Invitrogen) in MOPS SDS Running Buffer (life technologies) and transferred to a nitrocellulose membrane using the Bio-Rad Trans-Blot Turbo Transfer System. Blots were blocked in 5% bovine albumin serum (BSA, Sigma-Aldrich) in TBS-T buffer (Bio-Rad) and subsequently incubated in primary antibodies in 5% BSA in Tris-buffered Saline (Bio-Rad) with 0.1% Triton X-100 (Sigma-Aldrich) (TBS-T buffer) at 4 °C overnight. The next day, blots were incubated in secondary antibodies in 5% BSA. Enhanced luminol-based chemiluminescent (ECL) detection was performed using the Pierce™ ECL Western Blotting Substrate (Thermo Scientific). Images were processed and analyzed using ImageJ/FIJI. The following primary antibodies were used: anti-TSC2 (rabbit, Cell Signaling, 1:500), anti-pS6 (rabbit, 1:2000, Cell Signaling), anti-S6 (rabbit, Cell Signaling, 1:8000), horseradish peroxidase (HRP)-conjugated anti-Actin (mouse, Abcam ab49900, 1:25,000), and HRP-conjugated anti-rabbit (goat, Abcam ab6721, 1:8000).

The appropriate numbers of MDA-MB-436 cells were seeded on 6-well cell culture plates and left overnight to obtain optimal attachment to the surface. Subsequently, fresh AgNPs stock suspension (or medium without NPs for control plates) was added (10 or 50 μg/mL AgNPs). After 24 or 48 h, medium was aspired, cells were washed with PBS and trypsinized, collected and transferred into Eppendorf tubes. Then, cells were centrifuged (12,000× g for 10 min at 4 °C), the supernatant was discarded and the pellet was washed twice with PBS, then precipitated cells were frozen at −80 °C for further analysis. Cells lysates were prepared by suspending cells pellets in radioimmunoprecipitation assay buffer (RIPA) (Thermo Fisher Scientific Inc., Waltham, MA, USA) supplemented with a protease and phosphatase inhibitors cocktail.