Cell lysates were prepared in Pierce RIPA Buffer (Thermo Fisher Scientific, 89901) containing protease inhibitor and PhosStop cocktails (Roche, 5892970001 and 4906845001, respectively), separated on an SDS-polyacrylamide gel and transferred to a PVDF membrane and probed. The following primary antibodies and dilutions were used: Strep Tag (Thermo Scientific, MA517282, 1:1000) and β-actin-HRP (Cell Signaling, 5125, 1:2000). Images were captured using the Azure c600 Western Blot Imaging System.
Phosstop cocktail
Manufactured by Roche
Sourced in France
PhosSTOP is a phosphatase inhibitor cocktail for the inhibition of serine/threonine and tyrosine phosphatases in biological samples. It is designed to prevent the dephosphorylation of phosphoproteins during sample preparation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Thermo Fisher Scientific (6 mentions)
Complete protease inhibitor cocktail, by Roche (4 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Pierce ripa buffer, by Thermo Fisher Scientific (3 mentions)
Piercetm bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
β actin hrp, by Cell Signaling Technology (2 mentions)
Protease inhibitor and, by Roche (2 mentions)
Azure c600 western blot imaging system, by Azure Biosystems (2 mentions)
Strep tag, by Thermo Fisher Scientific (2 mentions)
Dimethyl sulfoxide (dmso), by Thermo Fisher Scientific (2 mentions)
Hek293f cell, by Thermo Fisher Scientific (2 mentions)
Anti phospho s6k1 t389 antibody, by Cell Signaling Technology (2 mentions)
Nupage lds sample loading buffer, by Thermo Fisher Scientific (2 mentions)
Anti ha antibody, by Santa Cruz Biotechnology (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Chemidoc mp system, by Bio-Rad (2 mentions)
Psd95, by Cell Signaling Technology (1 mentions)
Glun2b, by Proteintech (1 mentions)
Glun2a, by Proteintech (1 mentions)
19 protocols using phosstop cocktail
1
Protein Expression Analysis by Western Blot
2
Quantifying Synaptic Proteins in Tissues
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Antibodies against RAB39B (Cat# D-12162-1-AP), GluA2 (Cat# 11994-1-AP), GluN2A (Cat# 19953-1-AP), GluN2B (Cat# 19954-1-AP), and tyrosine hydroxylase (TH, Cat# 66334-1-IG) were from Proteintech. Antibodies against GFAP (Cat# 3670s), β-actin (Cat# 8457s), p62 (Cat# 5114s), GluN1 (Cat# 5704s), PSD95 (Cat# 3450s), LC3B (Cat# 3868s), S6 (Cat# 2217s), and phosphorylated S6 (Ser240/244) (Cat# 5364s) were from Cell Signaling Technology. Antibodies against GluA1 (Cat# 04-855), GluA3 (Cat# MAB5416), and SYN1 (Cat# AB1543) were from Millipore. Antibodies against NeuN (Cat# ab177487), Iba1 (Cat# 016-20001), and synaptophysin (SYP) (Cat# S5768) were from Abcam, Wako, and Sigma-Aldrich, respectively. Goat anti-rabbit (Cat# 31460) or anti-mouse (Cat# 31430) IgG (HCL) secondary antibodies conjugated with horseradish peroxidase were from Thermo Fisher Scientific.
The mTOR inhibitor rapamycin (Cat# HY-10219) was from MedChemExpress. DMSO (Cat# 20688) was from Thermo Fisher Scientific. 4′,6-diamidino-2-phenylindole (DAPI) (Cat# D9542) was from Sigma-Aldrich. Complete Protease Inhibitor and PhosSTOP Cocktails were from Roche.
Different protein levels in mouse tissues and cells were determined by western blot. Detailed procedures are presented inSupplementary Materials and Methods . Protein band intensity was quantified by densitometry using the Image J software (National Institutes of Health).
The mTOR inhibitor rapamycin (Cat# HY-10219) was from MedChemExpress. DMSO (Cat# 20688) was from Thermo Fisher Scientific. 4′,6-diamidino-2-phenylindole (DAPI) (Cat# D9542) was from Sigma-Aldrich. Complete Protease Inhibitor and PhosSTOP Cocktails were from Roche.
Different protein levels in mouse tissues and cells were determined by western blot. Detailed procedures are presented in
3
Western Blot Analysis of Cellular Proteins
4
Immunoprecipitation of CD20, ORAI-1, and IGF2R
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Total protein extracts were prepared in 1% Nonidet P‐40 detergent buffer (20 mM Tris pH 8, 137 mM NaCl, 2 mM EDTA, 10% glycerol and cOmplete® and PhosSTOP® cocktails, Roche). The antibodies used for immunoprecipitation of anti‐CD20 (sc‐7736 M‐20, Santa Cruz), anti‐ORAI‐1 (ab59330, Abcam) and anti‐IGF2R (sc‐14413 K21, Santa Cruz) were cross‐linked to 25 μl of Dynabeads Protein G (Thermo Fisher) in accordance with the manufacturer's instructions. Total protein lysates (1,000–1,500 μg) were incubated with the appropriate antibody–Dynabeads complexes and mixed ON at 4°C. The immune complexes were washed three times in 1 × PBS and the proteins were eluted boiling the complexes in 2 × Laemmli buffer (Tris 0.5 M pH 6.8; SDS 2%; glycerol 20%; β‐mercaptoethanol; bromophenol blue) for 10 min. Next, the samples were centrifuged at 10,000 × g for 5 min, and the resulting supernatants (IP samples) were collected.
5
Platelet GPIbα Immunoprecipitation and Analysis
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Resting platelet whole-cell lysates were prepared using RIPA buffer65 (link) containing protease (cOmplete) and phosphatase inhibitor (PhosSTOP) cocktails (Roche, Germany). GPIbα was immunoprecipitated from whole-cell lysates (0.2–0.5 mg) using Xia-B2 monoclonal antibody or isotype-matched control immunoglobin G (5 μg) for 1 h, followed by addition of 20 μl protein A/G Plus overnight at 4 °C. SDS–polyacrylamide gel electrophoresis (PAGE) and immunoblot analysis were performed26 (link)68 (link), following GPIbα immunoprecipitation, whereby samples were mixed with SDS sample buffer in the presence of 0.1 M dithiothreitol, and heated to 95 °C for 5 min. Equal protein concentration for each sample were subjected to SDS–PAGE on standard 12.5% (v/v) polyacrylamide gel, transferred to polyvinylidene difluoride (PVDF) and probed for GPIb with RAM-6 mAb (1 μg μl−1; ref. 69 (link)) or anti pan-14-3-3 pAb (0.2 μg ml−1), followed by horseradish peroxidase (HRP)-conjugated secondary antibody (1:10,000 from the stock solution) and enhanced chemiluminescence.
6
Protein Expression Analysis by Western Blot
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Cell lysates were prepared in Pierce RIPA Buffer (Thermo Fisher Scientific, 89901) containing protease inhibitor and PhosStop cocktails (Roche, 5892970001 and 4906845001, respectively), separated on an SDS-polyacrylamide gel and transferred to a PVDF membrane and probed. The following primary antibodies and dilutions were used: Strep Tag (Thermo Scientific, MA517282, 1:1000) and β-actin-HRP (Cell Signaling, 5125, 1:2000). Images were captured using the Azure c600 Western Blot Imaging System.
7
Western Blot Analysis Protocol
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Cells were lysed in RIPA buffer (Thermo Fisher Scientific) containing protease inhibitor and phosphatase inhibitor (PhosStop) cocktails (Roche). Protein extracts were quantified by Bradford (Bio-Rad) and equal amounts subjected to SDS-PAGE (4%–12%; Bio-Rad or Invitrogen). Proteins were transferred to a PVDF or a nitrocellulose membrane using a semidry transfer method. Blots were blocked using TBS-T (0.1% Tween 20) with 3% fat-free milk and 1% BSA and incubated with primary antibodies (listed in Supplemental Table S7 with dilutions in TBST with 1%BSA) overnight at 4°C. Horseradish peroxidase (HRP)-conjugated or fluorophore-conjugated antibodies were used as secondary antibodies (Supplemental Table S7 ) in TBST with 1% BSA for 1 h at room temperature and detected by a ImageQuant LAS400 system or ChemiDoc MP imaging system (Bio-Rad), respectively. Signals were quantified using the Fiji-ImageJ gel analysis software.
8
HA-S6K1 Kinase Mutant Analysis
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HA-S6K1ki wild type and mutants were cloned into the pcDNA3.1(+) vector. HEK-293F cells (Invitrogen) were maintained in DMEM medium with 10% fetal bovine serum (Sigma) at 37°C and 5% CO2. For transfection, cells were seeded into 6-well tissue culture plates, cultured to 70% confluence and exchanged into fresh media one hour prior to transfection. Cells were transfected with 2 μg each of HA-S6K1ki wild type or mutant plasmids using Lipofectamine 2000 (Invitrogen). 48 hours post transfection, cells were lysed in 50 mM Tris-HCl, 150 mM NaCl, 0.5 mM TCEP, 1% Triton-X100, 2 mM EDTA, 50 mM NaF, 10 mM β-glycerolphosphate and 10 mM Na-pyrophosphate, pH 7.5, and 1 tablet each of cOmplete protease inhibitor and PhosSTOP cocktail (Roche). Whole cell extract (W.C.E.) were adjusted to 2 mg ml−1 with lysis buffer and NuPAGE LDS sample loading buffer (Invitrogen), and boiled for 5 minutes. 20 μg W.C.E. were loaded on gel for immunoblotting with anti-HA antibody (Santa Cruz, SC805) or anti-phospho-S6K1 (T389) antibody (Cell signaling, #9205). The immunoblots were quantified by normalizing the anti-phospho-S6K1 signal to the anti-HA signal of each reaction.
9
Western Blot Analysis of P-SMAD1/5/9
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For detection of the P‐SMAD1/5/9 protein level, cells were washed twice with PBS and lysed in 1xRIPA buffer (50 mM Tris HCl pH 7.5, 150 mM NaCl, 1% Nonidet P‐40, 1% Sodium Deoxycholic, 0.1% SDS), containing phosphatase and protease inhibitors (PhosSTOP cocktail and Complete tablets, Roche), NaF 50 mM and Na3VO4 1 mM. Protein concentration was determined by the PierceTM BCA Protein Assay Kit (Thermo Scientific) according to the manufacturer's protocol and 15 µg of total lysates run onto precasted 4%–15% Midi Criterion TGX‐gels 18W (BioRad). Proteins were transferred onto Nitrocellulose membrane (BioRad) and probed with the indicated primary antibody at 4℃ overnight. After incubation with HRP‐conjugated secondary antibodies, protein bands were revealed by chemiluminescence with the Amersham ECL Detection Reagents (GE Healthcare) and detected with the Uvitec instrument (Cambridge, UK). Densitometric analysis of the western blot signals was performed by using the Uvitec software. Primary antibodies were diluted as follows: P‐SMAD1/5/9 1:2000 (#13820, SMAD1 1:2000 #9743, Cell Signaling); anti‐FLAG M2 monoclonal antibody 1:3000 (#F1804, Sigma‐Aldrich, Merk); anti‐GAPDH 1:20000 (#MAB374, Millipore).
10
Co-Immunoprecipitation Assay for PPK1:CFP Bait
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For co-IP using PPK1:CFP as bait, collected tissues were ground in liquid nitrogen. Proteins were then extracted into MOPS buffer (100 mM MOPS, pH7.6, 150 mM NaCl, 0.1% NP40, 1% Triton X-100, 20 mM Iodoacetamide, 1 mM phenylmethylsulfonyl fluoride, 2 μg l−1 aprotinin, 5 μg l−1 Leupeptin, 1 μg l−1 pepstatin, 2 × Complete protease inhibitor Cocktail and PhosStop cocktail from Roche), centrifuged and filtered through two layers of miracloth, precleared with protein A agarose beads, and then incubated with in-house rabbit anti-GFP antibodies for 1 h at 4 °C. PPK1:CFP fusion-proteins were then captured with protein A agarose beads for another hour at 4 °C, washed five times with IP buffer, transferred into a new tube before elution with boiling SDS sample buffer. The co-IP experiment was repeated three times.
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