Cells were harvested, washed with PBS, and lysed in RIPA buffer (150 mM NaCl, 50 mM Tris-HCl, 0.1% SDS, 1% NP-40, and 1% Triton X-100) supplemented with 1 mM PMSF (Sigma) and a protease inhibitor cocktail (Roche) at 4°C for 20 min. The lysates were then centrifuged for 15 min at 12,000 rpm at 4°C. The supernatants were collected, and an equal volume of 2X Laemmli’s buffer was added. The sample was boiled for 5 min at 95°C. Proteins were resolved by 10 or 12.5% SDS-PAGE and then transferred to nitrocellulose membranes (Pall Corporation). Membranes were blocked with 5% non-fat milk in TBST (0.1% Tween 20) for 1 h before incubation with primary and secondary antibodies sequentially. Signals were detected using SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific) according to the manufacturer’s instructions. The following antibodies were used: rabbit anti-FOP (Abcam, ab156013, 1:2,000), mouse anti-GFP (Santa Cruz, sc-9996, 1:5,000), rabbit anti-AURKA (Cell signaling Technology, 14475, 1:2,000), rabbit anti-Cyclin A2 (Abcam, ab18159, 1:10,000), rabbit anti-pCDC2 (Tyr15) (Cell Signaling Technology, 9111, 1:2,000), rabbit anti-pRb (Ser807/811) (Cell Signaling Technology, 8516, 1:2,000), and mouse anti-β-actin (Sigma, A5441, 1:5,000).
Sc 9996
Manufactured by Cell Signaling Technology
Sc-9996 is a laboratory reagent manufactured by Cell Signaling Technology. It is used for research purposes.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Pall Corporation (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (1 mentions)
Mouse anti β actin, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Rabbit anti prb ser807 811, by Cell Signaling Technology (1 mentions)
Rabbit anti aurka, by Cell Signaling Technology (1 mentions)
Rabbit anti cyclin a2, by Abcam (1 mentions)
Rabbit anti pcdc2 tyr15, by Cell Signaling Technology (1 mentions)
Bafa1, by Thermo Fisher Scientific (1 mentions)
Cycloheximide (chx), by Merck Group (1 mentions)
Hoechst, by Thermo Fisher Scientific (1 mentions)
Fibronectin, by Merck Group (1 mentions)
Epoxomicin, by Merck Group (1 mentions)
Doxycycline, by Merck Group (1 mentions)
Snap cell tmr star, by New England Biolabs (1 mentions)
Ab21685, by Abcam (1 mentions)
Ab24586, by Abcam (1 mentions)
Oregon green, by New England Biolabs (1 mentions)
Dsp crosslinker, by Thermo Fisher Scientific (1 mentions)
5 protocols using sc 9996
1
Western Blot Analysis of Cell Signaling Proteins
2
Molecular Probes for Organelle Dynamics
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Reagents and antibodies were purchased from the following suppliers: anti-Giantin (Abcam; ab24586, 1:1000), anti-BiP for immunofluorescence (Abcam ab21685, 1:500), anti-BiP for Western blot (Cell signaling, 3183S, 1:1000), anti-KDEL (Abcam ab176333, 1:1000), anti-GFP (Santa Cruz sc-9996, 1:1000), anti-HA (Cell signaling 3724, 1:1000), anti-GAPDH (Cell signaling 2118, 1:2000), TMP (Sigma), CHX (Sigma), doxycycline (Sigma), fibronectin (EMD Millipore), Hoechst (Thermo Fisher Scientific; 10 mg/ml in H2O, 1:2000) D/D solubilizer (Clonetech), BafA1 (Alfa Aesar), epoxomicin (EMD Millipore), SNAP-tag ligands (SNAP Cell TMR Star and Oregon Green [New England Biolabs]), HT TMR ligand (Promega), and DSP Crosslinker (Thermo Fisher Scientific). HyT36 and HyT36(-Cl) were previously described (Tae et al., 2012 (link); Serebrenik et al., 2018 (link)).
3
Western Blot Analysis of Autophagy Markers
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For western blot analysis, cells were harvested in 70 μL lysis buffer (3% SDS, 30 mM Tris base, pH 8.8, 5 mM EDTA, 30 mM NaF, 10% glycerol, 1 mM DTT) and 25–50 μg protein with 6x Laemmli buffer was loaded onto 4–20% Bio-Rad Mini- PROTEAN Precast Gels or 12% self-casted acrylamide/bisacrylamide (29:1) gels. After electrophoresis, the proteins were transferred to PVDF membranes with Bio-Rad Tris/Glycine buffer or self-made transfer buffer (25 mM Tris base, 190 mM glycine, 20% methanol, pH 8.3) and stained with the following antibodies: ubiquitin – FK2 clone Millipore, 04–263; mCherry – Novus Biologicals, NBP1-96,752; GFP – Santa Cruz Biotechnology, sc-9996; LC3B – Cell Signaling Technology, 2775; SQSTM1/p62 – Sigma-Aldrich, P0067; ZFTVE1/DFCP1 – Cell Signaling Technology; WIPI2 – Invitrogen; ACTN2 (actinin alpha 2) – Sigma-Aldrich, A2543 or A7811; RPS6 (ribosomal protein S6) – Cell Signaling Technology, 2771; GAPDH – HyTest, 5G4. Western blots were either detected with LI-COR Biosciences Odyssey (Figures 4A, E, G, M and 5D ) or Biorad ChemiDoc (Figures 4C, I, J, K and 5E ).
4
Biochemical Characterization of Akt Signaling
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Chemical agents employed in the studies include the pan-PI3K inhibitor PI-103 (Cayman Chemical), Akt inhibitor VIII (Akti; Millipore), and the diC8 phosphoinositides PI(3,4,5)P3, phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) from Echelon Biosciences. The antibodies used targeted GFP (Santa Cruz sc-9996), phospho-Ser/Thr Akt substrate sites (Cell Signaling 9614), Akt phospho-Thr308 (Cell Signaling 9275), or HCN2 (Abcam ab19346). Active recombinant Akt1 protein was from Millipore.
5
Western Blot Analysis of Cellular Proteins
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Protein extracts were prepared in Laemmli loading buffer containing 2.5% beta-mercaptoethanol (except for experiment in non-reducing condition where no beta-mercaptoethanol was added) and heated for 5 min at 95°C before separation of 20 μg total proteins on 8 or 12% SDS-PAGE gels. Separated proteins were transferred onto nitrocellulose or PVDF membranes. The membranes were blocked in TBS (Tris-buffered saline; 50 mM Tris-Cl, pH 7.6, 150 mM NaCl), 0.1% Tween-20, 5% non-fat milk. The following antibodies were used: anti-GFP (Santa Cruz sc-9996, 1:1,000), anti-GABARAP/Atg8a (Cell Signaling Technology No. 13733, 1:2,000), anti-Ref(2)P (Abcam ab178440, 1:1,000), anti-β actin (Abcam ab8227, 1:2,000), anti-α tubulin (Sigma-Aldrich T5168, 1:40,000), HRP-coupled secondary antibodies anti-rabbit and anti-mouse (Thermo Scientific No. 31460 and 31450, 1:10,000). Signals were developed using the ECL detection reagents (Amersham, RPN2209).
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