Phosphatase inhibitor cocktail

Cell membranes were lysed in buffer A containing 10 mM KCl, 2 mM MgCl₂, 1 mM DTT, 0.1 mM EDTA, 10 mM HEPES, pH 7.8, protease inhibitor cocktail (Roche), and phosphatase inhibitor cocktail (Roche) for 10 min on ice. The lysis solution was centrifuged at 4 °C and 14,000 rpm for 15 min. The supernatant (cytoplasmic components) was moved to a new Eppendorf 1.5 mL tube and the pellet (nuclear components) was washed two times with buffer A before the nuclear envelope was lysed using ultrasound in buffer C, which contained 1 mM KCl, 1 mM DTT, 0.1 mM EDTA, 50 mM HEPES, pH 7.8, 300 mM NaCl, 20% glycerol, protease inhibitor cocktail (Roche), and phosphatase inhibitor cocktail (Roche). The nuclear proteins were collected by centrifuging at 4 °C and 14,000 rpm for 15 min.

For protein extraction from S2R+ cells, cell lysates were extracted in lysis buffer (50 mM TrisCl, pH 7.6, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100) with protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktail (Roche). For protein extraction from adult flies, one-day-old flies were collected, and proteins were extracted in lysis buffer with protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktail (Roche). Protein concentrations of the cleared lysate after two times centrifugation (13,000 r.p.m.) at 4 °C were measured, and the same amount of proteins was used for western blot analysis.
For immunostaining of western blots, following antibodies were used: mouse anti-V5 (1:5000, Invitrogen R960-25), mouse anti-Myc (1:1000, Santa Cruz sc-47694), rabbit anti-CCT4 (1:20,000) (this study), rat anti-CCT1 antibody (1:1000, Abcam ab90357), rat anti-Rheb (1:500) [48 (link)], and mouse S6K (1:1000) [40 (link)], rabbit phospho-S6K (1:1000, Cell signaling 9209), rabbit phospho-S6 (1:5000) [49 (link)], rabbit anti-Histone 3 (1:10,000, Millipore 05-928), rabbit anti-GAPDH (1:5000, GeneTex 100118), rabbit anti-Akt (1:1000, Cell Signaling 4691), rabbit anti-phospho-Akt (1:1000, Cell Signaling 4054), mouse anti-Tubulin (1:5000, Sigma T5168), guinea pig anti-TOR (1:2000) [50 (link)], rat anti-TOR (1:2000) [51 (link)].

Male mice (mean age of 12 weeks) were anesthetized by intraperitoneal injection of PBS (137 mM NaCl, 8.1 mM Na₂HPO₄, 2.68 mM KCl, 1.47 mM KH₂PO₄, pH 7.4) supplemented with ketamine (20 mg/mL) and xylazine (4 mg/mL). Animals were then transcardially perfused with PBS containing 0.25% phosphatase inhibitor cocktail, 1% protease inhibitor (Roche) and 20 U/mL heparin. Perfused brain, spinal cord, liver, and quadriceps muscle were collected, frozen on dry ice and stored at -80°C until further processing.
For western blot analysis, enzyme-linked immunosorbent assay (ELISA) and protein oxidation detection, tissues were mechanically homogenized in 150–200 μL homogenizing buffer [PBS containing 1:100 protease inhibitor cocktail (Roche), 1:50 phosphatase inhibitor cocktail and 1:20 DNase (Roche)] before ultrasonication at 2 amp for 10 s. Homogenates were centrifuged at 18,000 g for 3 min at 4°C and the supernatant (soluble fraction) collected and the pellet (insoluble fraction) retained. Protein content was determined by the bicinchoninic acid assay (Thermo Scientific, USA). Fractions were stored at -80°C until further analysis.

Transfected 293T and HeLa cells were lysed with CelLytic^™ M supplemented with a complete protease inhibitor cocktail (Roche Applied Science) and a phosphatase inhibitor cocktail (Roche Applied Science). Cell extracts were incubated with anti‐FLAG^® M2 affinity gel or anti‐HA‐agarose overnight. After the beads were washed three times with PBS, proteins bound to the beads were eluted by elution buffer (3X FLAG^® peptide (Sigma‐Aldrich) or HA peptide (Sigma‐Aldrich) in PBS) containing a complete protease inhibitor cocktail (Roche Applied Science) and a phosphatase inhibitor cocktail (Roche Applied Science). Eluted samples were boiled with Lane Marker Sample Buffer (Thermo Fisher Scientific), and used for western blot analysis.

Cells were lysed in lysis buffer (20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 50 mM NaF, 0.5% NP-40, 1 mM EDTA, 100 mM N-ethylmaleimide [NEM]) containing a complete protease inhibitor cocktail (EDTA-free; Roche) and a phosphatase inhibitor cocktail (Roche). For denaturing IP, cells were lysed in 1% SDS/radioimmunoprecipitation assay [RIPA] buffer (25 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 100 mM NEM) containing a complete protease inhibitor cocktail (EDTA-free; Roche) and a phosphatase inhibitor cocktail (Roche). SDS was diluted to 0.1% prior to IP reactions. Anti-DYKDDDDK-tag mAb-agarose (M185-11; MBL) and anti-p62 rabbit polyclonal (PM045; MBL) were used to precipitate the target protein complex by incubation for 2 h at 4°C. After three washes with lysis buffer or 0.1% SDS/RIPA buffer, coimmunoprecipitates were subjected to downstream analyses.

Cells (HEK293T, U2OS) were lysed in NP‐40 lysis buffer (50 mM Tris, pH7.5, 120 mM NaCl, 1% NP‐40) supplemented with Complete^® protease inhibitor (Roche) and phosphatase inhibitor cocktail (Roche). Lysates were passed through a 27G needle, centrifuged at 21,000× g and incubated with either anti‐GFP agarose (Chromotek, gta‐20) or anti‐HA agarose (Sigma, A2095), washed three times in lysis buffer and subjected to SDS–PAGE and Western blot. For total cell lysis (TCL), cells were lysed in 50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM MgCl₂, 1% SDS. TCL buffer was supplemented with Complete^® protease inhibitor (Roche), phosphatase inhibitor cocktail (Roche) and Benzonase (VWR/Fisher Scientific) at 1 μl per ml buffer. Samples were boiled in 3× Laemelli buffer prior to SDS–PAGE. Unless otherwise stated, NuPAGE™ 4–12% Bis–Tris gradient gels (Invitrogen) were used. Gels were transferred onto activated PVDF membranes (Immobilon‐Psq, 0.2 μm, Merck) prior to blocking and incubation with the indicated primary antibodies.

Deoxycholate fraction of fibronectin was conducted according to the previously published protocol^{25 (link)}. Briefly, cells attached on 2D surface or collected from 3D culture plate were washed with ice-cold PBS, followed by being lysed with DOC buffer (2% Deoxycholate, 0.02 M Tris-HCl,pH 8.8, 2 mM PMSF, 2 mM NaF, 2 mM Na7VO4, phosphatase inhibitor cocktail (Roche), protease inhibitor cocktail (Sigma)). Lysis was carried out by passing the lysate through a 23-gauge syringe needle more than ten times, followed by incubation on rotor wheel for 30 minutes at 4 °C. The lysates were centrifuged at 13,000 × g for 30 min at 4 °C, the supernatants were transferred to other tubes as the soluble fraction. Pellets were washed briefly with DOC buffer and the used buffer was discarded, remaining pellet was designated as the insoluble fraction. The insoluble fraction was resuspended by 2× laemmli sample buffer (0.25 M Tis-HCl, 20% glycerol, 4% Sodium dodecyl sulfate, 0.004% Bromophenol blue, phosphatase inhibitor cocktail (Roche), protease inhibitor cocktail (sigma)). The soluble fraction was mixed with 2× laemmli sample buffer and used for the internal comparison of ratio among samples.