Ip lysis wash buffer

HEK293 cells transiently transfected with indicated plasmids and harvested using ice-cold IP Lysis/Wash Buffer (Thermo-Fisher) including 20 mM N-ethylmaleimide and protease inhibitor cocktail. Protein concentrations were determined in a Direct Detect Spectrometer (Merck Millipore). Co-immunoprecipitation (Co-IP) was performed using a V5-antibody (Thermo-Fisher; #R960-25) and the Pierce Co-IP kit (Thermo-Fisher). Samples were analyzed by SDS-PAGE and immunoblotting with mouse anti-V5 (Thermo-Fisher), rabbit anti-HA (Cell Signaling; #3724), rabbit anti-Flag (Cell Signaling; #2368) and horseradish peroxidase (HRP)-linked secondary antibodies.

Cells were lysed with IP Lysis/Wash Buffer (Thermo Scientific). The lysates were collected using a scraper and centrifuged at 13,000 RPM for 10 min and the supernatant was transferred to a new tube. The protein concentration of the cell extract was determined using a BCA Protein Assay Kit. Equal amounts of each protein sample (~500 μg/lane) were incubated with anti-SIRT1 and ubiquitin monoclonal antibodies (1:500) at 4° C, with overnight shaking. The immunocomplexes were incubated with Pierce Protein A/G Agarose for 1 h. After washing, the samples were precipitated with conditioning buffer, followed by immunoblot analysis.

Glioma cells were treated with amlexanox (50, 100 and 150 μM) for 72 h or treated with 150 μM amlexanox for 24, 48 and 72 h. Cultured cells were lysed in RIPA buffer supplemented with protease inhibitor. The protein concentrations were evaluated with a BCA protein assay kit (Pierce, USA). The samples were heated in 100 °C water for 10 min to denature the proteins, separated on 10 or 12% SDS-PAGE gels, and electrophoretically transferred to a PVDF membrane (Millipore, USA). For the co-immunoprecipitation (Co-IP) experiments, glioma cells were lysed in IP Lysis/Wash Buffer (Thermo, USA). Fifty microliters of protein G agarose (Santa Cruz Biotechnology, CA, USA) was mixed with a specific monoclonal antibody or a nonspecific IgG overnight at 4 °C. The immunoprecipitated proteins were separated by SDS-PAGE and analyzed by western blot. For endogenous LATS2 ubiquitination assay, Lysates were incubated using 2 μg anti-LATS2 antibody and analyzed by western blot using anti-Ubiquitin or anti-LATS2 antibodies.

The deacetylase activity of SIRT1 was determined by using a SIRT1 Activity Assay Kit (Abcam, ab156065) as recommended by the manufacturer. Briefly, cells were washed with cold PBS, lysed in IP Lysis/Wash buffer (Thermo Fisher) and incubated on ice for 5 min. After two homogenization cycles (7 s) with an ultrasonic cell disruptor (Microson; Misonix), total cell lysates were centrifuged 10 min at 13,000g, and supernatant containing proteins was collected. The protein concentration of the extracts was determined by Pierce BCA Protein Assay Kit (Thermo Scientific). Cell lysates (200 μg) were incubated with anti-SIRT1 antibody (10 μg) (Abcam, ab7343) for 3 h at 4 °C and then with protein A Agarose beads for the next 1.5 h. Precipitates were incubated with Fluoro-Substrate Peptide Solution, NAD⁺ and SIRT1 Assay Buffer. Fluorescence intensity was then measured using a microtitre plate fluorometer with excitation at 360 nm and emission at 460 nm.

Cells were collected and lysed with RIPA buffer (Beyotime, P0013C, Shanghai, China) after being treated with the indicated drugs. Then, the supernatant was collected, and the total protein concentration of the cells was determined with a BCA kit (Beyotime, P0010, Shanghai, China). After separation by 10% SDS-PAGE (Beyotime, P0014A, Shanghai, China), the samples were electrotransferred to a polyvinylidene fluoride membrane (Millipore, ISEQ00010, Billerica, MA, USA). After being treated with the specific primary antibody, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 1–2 h. They were washed with TBST three times. Then, the bound antibody complexes were examined by enhanced chemiluminescence solution (ECL, Amersham Pharmacia Biotech, Piscataway, NJ, USA) and analysed by a ChemiDoc XRS + system (BioRad, USA).
For co-immunoprecipitation (Co-IP) experiments, A549 and A549/DDP cells were lysed in IP Lysis/Wash Buffer (Thermo, 88805, USA). Then, 25 µL of protein A/G magnetic beads were mixed with a specific monoclonal antibody or nonspecific IgG overnight at 4 °C. The immunoprecipitated proteins were separated by SDS-PAGE and analysed by western blot analysis.

Cells were lysed in IP lysis/wash buffer (Thermo‐scientific) without the addition of protease and phosphatase inhibitors. For glycosylation analysis, 5 μg OPC lysates were then subjected to digestion for 1 hr at 37 with EndoH enzyme or EndoH glycobuffer (NEB) alone as control. For phosphorylation analysis, 5 μg of OPC lysate were subjected to digestion for 30 min at 30 with Lambda protein phosphatase or 10× NeBuffer Protein MetalloPhosphatases (PMP) and MnCl₂ only as control. The OPC lysates were then boiled for 10 min at 95 and further processed for Western Blot as described above.