Whole cell lysates were prepared with lysis buffer. Extracts were resolved on SDS-PAGE, and transferred to nitrocellulose membrane (Whatman, Scheicher & Schuell, Dassel, Germany). Membranes were incubated with the specific antibodies overnight at 4°C, washed and incubated with the appropriate secondary antibody, for 1 h at room temperature. Antibodies were used against: pAKT (s473) #9271, pS6R (s235/236) #2211, pMEK (s217/221) #9121, LC3B(D11) #3868, pBRAF #2996, total Caspase-3 #9662, cleaved caspase-3 #9661 from Cell Signalling (Danvers, MA, USA), BECN1 (sc-10086), SQSTM1/p62 (sc-28359), PARP (sc-7150), pERK (sc-7583), and Tubulin (sc-8035) from Santa Cruz (Biotechnology, Inc. 2145 Delaware Avenue Santa Cruz, CA 95060 USA). Antibody signal was obtained with the enhanced chemiluminescence plus Western blotting detection system (Amersham Biosciences, Uppsala, Sweden) after exposure to Kodak Super RX film. Values were measured using the Image-Quant software (Amersham Biosciences) and protein levels were normalized against tubulin. Experiments were independently repeated three times and standard deviation is presented.
Tubulin sc 8035
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Tubulin (sc-8035) is a protein that is a major component of microtubules, which are cytoskeletal structures involved in a variety of cellular processes. It is used for research purposes in cell biology and biochemistry applications.
Automatically generated - may contain errors
Lab products found in correlation
Total caspase 3, by Cell Signaling Technology (1 mentions)
Enhanced chemiluminescence plus western blotting detection system, by Cytiva (1 mentions)
Lc3b d11, by Cell Signaling Technology (1 mentions)
Imagequant software, by Cytiva (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Nigericin, by Merck Group (1 mentions)
Lamin b, by Cell Signaling Technology (1 mentions)
Rabbit igg sc 2027, by Santa Cruz Biotechnology (1 mentions)
Sytox blue dead cell stain, by Thermo Fisher Scientific (1 mentions)
Z leu leu leu al mg132, by Merck Group (1 mentions)
Sytox red dead cell stain, by Thermo Fisher Scientific (1 mentions)
Zvad fmk zvad, by Merck Group (1 mentions)
Ha hrp 3f10, by Roche (1 mentions)
Pepstatin a, by Cayman Chemical (1 mentions)
Mitosox red mitochondrial superoxide indicator m36008, by Thermo Fisher Scientific (1 mentions)
P62 5114, by Cell Signaling Technology (1 mentions)
Aif 5318, by Cell Signaling Technology (1 mentions)
Flag hrp a8592, by Merck Group (1 mentions)
Ub p4d1, by Santa Cruz Biotechnology (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
4 protocols using tubulin sc 8035
1
Western Blot Analysis of Cell Signaling
2
Antibody and Reagent Protocols for Cell Death Analysis
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Antibodies for PARP (9532, 1:1000), ULK1 (8054, 1:1000), p62 (5114, 1:1000), AIF (5318, 1:1000) and Lamin B (12586, 1:1000) were purchased from Cell Signaling Technology (MA, USA). TFG (ab156866, 1:10,000) and GSDMD (ab210070, 1:1000) were from Abcam. Caspase-1 (AG-20B-0048-C100, 1:1000) were purchased from AdipoGen (CA, USA). HA (12CA5, 1:1000 for IP) and HA-HRP (3F10, 1:5000) were from Roche Life Science (Switzerland). Flag (F3165, 1:500 for IP) and Flag-HRP (A8592, 1:5000), LC3B (L7543, 1:1000) and β-actin (A2228, 1:10,000) were from Sigma–Aldrich (MO, USA). Ub (P4D1, 1:1000), TRAF3 (H-122, 1:1000), Tubulin (SC-8035, 1:1000), and rabbit IgG (sc-2027, 1:1000) were from Santa Cruz Biotechnology (CA, USA). COX4 (ARG66326, 1:1000) were purchased from Arigo Biolaboratories. V5 (R96025, 1:500 for IP) and V5-HRP (R96125, 1:5000) were from Thermo Fisher Scientific (MA, USA).
MitoSox™ Red Mitochondrial Superoxide Indicator (M36008), SYTOX™ Blue Dead Cell Stain (S34857), and SYTOX™ Red Dead Cell Stain (S34859) were purchased from Thermo Fisher Scientific (MA, USA). LPS, Nigericin, Z-VAD-FMK (Z-VAD), Z-Leu-Leu-Leu-al (MG132) were purchased from Sigma–Aldrich (MO, USA). Necrostatin-1 (NEC-1) was purchased from ApexBio (Texas, USA). Cycloheximide (CHX), E-64d, and Pepstatin A were purchased from Cayman Chemical (Michigan, USA).
MitoSox™ Red Mitochondrial Superoxide Indicator (M36008), SYTOX™ Blue Dead Cell Stain (S34857), and SYTOX™ Red Dead Cell Stain (S34859) were purchased from Thermo Fisher Scientific (MA, USA). LPS, Nigericin, Z-VAD-FMK (Z-VAD), Z-Leu-Leu-Leu-al (MG132) were purchased from Sigma–Aldrich (MO, USA). Necrostatin-1 (NEC-1) was purchased from ApexBio (Texas, USA). Cycloheximide (CHX), E-64d, and Pepstatin A were purchased from Cayman Chemical (Michigan, USA).
3
AMPK Pathway Regulation by Western Blot
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Cells were lysed for 30 min on ice in IP buffer with protease inhibitors cocktail (SRE0055-1BO, Sigma-Aldrich). Samples were quantified with BCA assays (23225, Thermofisher) and analyzed by western blotting. For IP, cell lysates were incubated with protein-specific antibodies or normal rabbit IgG (sc-2027, Santa Cruz Biotechnology, Dallas, TX, USA) overnight at 4 °C, followed by incubation with protein A/G agarose beads (Millipore; IP10) for 3 h and washed in lysis buffer supplemented with protease inhibitors. Antibodies for western blotting were: AMPK (SAB4502329), p-AMPK (Thr172, SAB4503754), Ki-67 (SAB5500134), Actin (A5441) (Sigma-Aldrich), tubulin (SC-8035, Santa Cruz, Dallas, TX, USA), PKM2 (4053, Cell Signaling, Danvers, MA, USA), H3 (ab1791), β-Catenin (ab32572) (Abcam, Cambridge, UK).
4
Western Blot Analysis of MAPK Signaling
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Cells were harvested and proteins were extracted using cell lysis buffer supplemented with 0.3% phenylmethylsulfonyl fluoride and proteinase and phosphatase inhibitors. Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). After blocking in 5% skimmed milk powder for 2 hours at room temperature, the samples were incubated overnight at 4°C with primary antibodies, including ERK1/2 (#9102), p-ERK1/2 (#9101), JNK1/2 (#9252), p-JNK (#9251), p38 MAPK (#9212), and p-p38 MAPK (#9211), which were obtained from Cell Signaling Technology (Beverly, MA, USA), as well as VEGF-a (sc-7269) and Tubulin (sc-8035), which were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). After washing three times with TBST (Tris-buffered saline supplemented with 0.1% Tween-20), the samples were incubated for 1 hour at room temperature with a secondary antibody. Bound antibodies were detected using the enhanced chemiluminescent substrate (ECL, Pierce, Rockford, IL, USA). The bands were assessed by densitometry with Image J software (National Institutes of Health).
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