10–20 µg of total protein was separated with 7.5–12% SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad). The membranes were blocked with 5% non-fat milk (LabScientific, Highlands, New Jersey) in TBS for 30 min and then incubated with primary antibodies in TBS containing 5% milk and 0.1% Tween-20 overnight at 4°C. After rinsing with TBS-0.1% Tween, membranes were incubated with the secondary antibodies diluted in 5% milk-TBS for 1.5 hr at RT. Bands were detected by using Odyssey CLx Scanner. Band intensity quantifications were performed using Image Studio 3.1. Normalizations were performed either relative to the actin or Akt band intensities. Western blots were performed with antibodies against p110α (#4249), p110β (#3011), Akt (#4691), phospho-Akt T308 (#13038), phospho-Akt S473 (#9271), phospho-Erk 1/2 T202/Y204 (#9101), phospho-S6 S235/236 (#2211), phospho-S6 S240/244 (#5364), phospho-EGFR Y1068 (#2236), S6 (#2217), HA (#2367), TfnR (transferrin receptor) (#13208), Nup (nucleoporin) (#2598), EGFR (#4267), PTEN (#9188) (all Cell Signaling), Rac1 (05–389) (Millipore, Billerica, Massachusetts), β-actin (A5441), α-tubulin (T5168) (Sigma), Caveolin1 (MA3-600) (Thermo Fisher) and Gq (sc-392) (Santa Cruz). IRDye 800CW Goat anti-mouse or anti-rabbit secondary antibodies (Li-Cor, Lincoln, Nebraska) were used.
α tubulin t5168
Manufactured by Merck Group
Sourced in United States, United Kingdom
α-tubulin (T5168) is a protein that is a component of the cytoskeleton in eukaryotic cells. It is commonly used in research as a structural marker for microtubules.
Automatically generated - may contain errors
Lab products found in correlation
β actin a5441, by Merck Group (3 mentions)
Donkey anti goat igg hrp sc 2020, by Santa Cruz Biotechnology (2 mentions)
Prolong gold antifade mountant with dapi, by Thermo Fisher Scientific (2 mentions)
A11008, by Thermo Fisher Scientific (2 mentions)
Pericentrin, by Merck Group (2 mentions)
Sp5 up, by Leica (2 mentions)
Hsc70 adi spa 815, by Enzo Life Sciences (2 mentions)
Nedd1, by Cell Signaling Technology (2 mentions)
A11004, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Rac1 05 389, by Merck Group (1 mentions)
Irdye 800cw goat anti mouse or anti rabbit secondary antibodies, by LI COR (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
P erk1 2 thr202 tyr204, by Cell Signaling Technology (1 mentions)
Cyclin d1, by Cell Signaling Technology (1 mentions)
P akt ser473, by Cell Signaling Technology (1 mentions)
Phistone h2a x, by Cell Signaling Technology (1 mentions)
Azure c400 imaging system, by Azure Biosystems (1 mentions)
Pan akt, by Cell Signaling Technology (1 mentions)
22 protocols using α tubulin t5168
1
Western Blot Analysis of PI3K/Akt Signaling
2
Western Blot Analysis of Cell Signaling
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Cell pellets were collected and lysed in RIPA buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, and other inhibitors, such as sodium orthovanadate, sodium fluoride, EDTA, and leupeptin. Protein samples were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore, MA, USA). The membrane was then blocked with 5% milk or 3% bovine serum albumin solution, followed by incubation with primary antibodies at 4 °C. The membranes were then washed three times with TBS containing 0.05% Tween 20 before being incubated with horseradish peroxidase-conjugated secondary antibody. The Azure c400 imaging system (Azure Biosystems, Inc., USA) was used for membrane exposure.
The following antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA), including p-Histone H2A.X (#2577), p53 (#9282 S), p21 (#2946), Akt (pan) (#2920), p-Akt (Ser 473) (#9271), p-Rb (Ser 807/811) (#9308), Cyclin D1 (#55506), Erk1/2 (#9102) and p-Erk1/2 (Thr202/Tyr204) (#9101). The p16INK4a (ab81278) antibody was obtained from Abcam PLC. (Cambridge, UK) and α-tubulin (T5168) was obtained from Sigma-Aldrich (St. Louis, MO, USA). GAPDH mouse mAb (E1A12400) was purchased from EnoGene Biotech Co., Ltd. (Nanjing, China).
The following antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA), including p-Histone H2A.X (#2577), p53 (#9282 S), p21 (#2946), Akt (pan) (#2920), p-Akt (Ser 473) (#9271), p-Rb (Ser 807/811) (#9308), Cyclin D1 (#55506), Erk1/2 (#9102) and p-Erk1/2 (Thr202/Tyr204) (#9101). The p16INK4a (ab81278) antibody was obtained from Abcam PLC. (Cambridge, UK) and α-tubulin (T5168) was obtained from Sigma-Aldrich (St. Louis, MO, USA). GAPDH mouse mAb (E1A12400) was purchased from EnoGene Biotech Co., Ltd. (Nanjing, China).
3
Antibody Characterization and Validation
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GFP (#sc-8834), p53 (DO-1, #sc-126) p53 FL-393 (s#c-6243), Myc (9E10, #sc-40) and normal rabbit IgG (sc-2027) antibodies were purchased from Santa Cruz Biotechnologies. Flag (M2, #F3165) and α-tubulin (#T5168) antibodies were obtained from Sigma. ACTIN (C4, #69100) antibodies were purchased from MP Biomedicals and HA (3F10, #11867423001) antibodies from Roche. Ubiquitin (P4D1, #3936), and p53 phospho-Ser46 (#2521) antibodies were from Cell Signaling Technology. Cleaved PARP (Y34, #ab32561) was obtained from Abcam. The H4 (#07-108) antibodies were from Millipore. The affinity-purified HIPK2 antibody and the chicken SIAH1 antibody have been described previously (22 (link)). Rabbit polyclonal DAZAP2 antibodies were raised against the following keyhole limpet hemocyanine (KLH)-coupled peptide: H2N-MNSKGQYPTQPTYPVC-COOH. The rabbit sera were affinity-purified against the peptide prior to use. The phosphorylation specific DAZAP2 Ser77 antibody was raised against the following phospho-peptide: H2N-YLPMA-S(p)-VAVGPLC-COOH and the phospho-specific antibodies were subsequently affinity purified.
4
Immunohistochemical analysis of TRPV1 and CGRP
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Antibodies used were TRPV1 (SC-12498) from Santa Cruz Biotechnology; CGRP (1720–9007) from Biogenesis; NF200 (N0142), and β-actin (A5441), and α-tubulin (T5168) from Sigma-Aldrich. The CPEB3 monoclonal and affinity-purified polyclonal antibodies were described previously [24 (link), 25 (link)]. Fura-2 acetoxymethyl ester (Fura 2-AM, F1221), Hoechst 33342 (H3570) and AlexaFluor-conjugated secondary antibodies, including Alexa 488-conjugated donkey anti-rabbit IgG (A21206), Alexa 594-conjugated donkey anti-goat (A11058) and donkey anti-mouse IgG (A21203), were from Invitrogen. DyLight 594-labled GSL (IB4, DL-1207) was from Vector Laboratories. CFA, (F5881), capsaicin (M2028) and type Ia collagenase (C9891) were from Sigma-Aldrich.
5
Immunoblotting Key Stress Signaling
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Primary antibodies were obtained from Cell Signaling [MKK4 #9152, MKK7 #4172, phospho- JNK1/2 (Thr183, Tyr185) #4668, p38 #9212, phospho-p38 (Thr180, Tyr182) #9211, and IκBα #4812, Danvers, MA, USA], BD Pharmingen (JNK1/2 #554285, Bedford, MA, USA), and Sigma (α-Tubulin #T5168, St. Louis, MO, USA).
6
Comprehensive Antibody Panel for Ciliary and Cytoskeletal Analysis
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The following antibodies were used in the study: acetylated α-tubulin (6–11-B-1, used at 1:10,000 to detect cilia and 1:1,000 to detect cytoplasmic microtubules) and α-tubulin (T5168, used at 1:1,000 for immunofluorescence (IF) and 1:50,000 for western blot) from Sigma; EB1 (610534, used at 1:200), β-catenin (610153, used at 1:200) and anti-PKAc (610980, used at 1:200) from BD biosciences; acetylated α-tubulin (ab24610, used at 1:500) and α-tubulin (ab18251, used at 1:500) from Abcam; IFT54 (HPA037858, Atlas Antibodies, used at 1:50 for IF and 1:1,000 for WB); ZO1 (61–7300, used at 1:100) from Life Technologies; ARL13B (17711-1-AP, Proteintech, used at 1:400); Gp135 (AF1556, R&D, used at 1:200 for IF and 1:1000 for WB); γ-tubulin (DQ-19, Sigma, used at 1:500); γ-tubulin (C-20, used at 1:200), MAP4 (H-300 and G-10, used at 1:400 for IF and 1:1,000 for WB) and ACIII (C-20, used at 1:200) from Santa Cruz; GAPDH (MAB374, used at 1:4000 for WB) from Millipore. Highly cross adsorbed secondary antibodies (Alexa Fluor 488, Alexa Fluor 546, AlexaFluor 555, AlexaFluor 532 and Alexa Fluor 647) were obtained from Molecular Probes (Life Technologies) and were used at 1:200 dilution.
7
Depletion of NonO and Adar1 with siRNAs
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NonO (L-048587-00-0005, 40 nM) (ON-TARGETplus smartpool siRNA, GE Dharmacon, USA) and Adar1 siRNA (L-048587-00-0005, 150 nM) (ON-TARGETplus smartpool siRNA, GE Dharmacon, USA) were used to deplete NonO and Adar1, respectively. The siRNAs were transfected to cells using Lipofectamine RNAiMAX reagent as per the manufacturer’s instructions (Invitrogen, USA) and incubated for 48 hrs. Knockdown was confirmed using NonO antibody (gift from Dr Yasuyki Kurihara, Yokohama National University, Yokohama, Japan)44 (link) and ADAR1 antibody (sc-73408; Santa Cruz Biotechnology, USA). Loading controls used were α-tubulin (T5168, SIGMA, USA) and B”-U2snRNP.
8
Immunoblot Analysis of Lipid Markers
9
Investigating Transcriptional Regulation in Cells
10
Immunoblot Analysis of Cellular Proteins
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Analysis of protein levels was carried out by immunoblot analysis of whole cell lysates using polyclonal antibodies against PARP1 (H-300, Santa Cruz), cyclin A (C-19, Santa Cruz), p53 (CM5, Novocastra, Newcastle, UK), p-p53 ser15 (9284, Cell Signaling), p38 (C-20, Santa Cruz), p-p38 (Thr180/Tyr182) (sc-17852, Santa Cruz), p16 (M-156, Santa Cruz), p-H2AX Ser139 (07-164 Upstate), p21 (M-19, Santa Cruz), PPM1D/WIP1 (H-300, Santa Cruz), and E2F1 (C-20, Santa Cruz), as well as monoclonal antibodies against pan-Ras-V12 (Ab-1, Calbiochem, Sigma-Aldrich, St. Louis, MO, USA), cyclin D1 (DCS6, Cell Signaling Technology, Inc., Danvers, MA, USA), α-tubulin (T5168, Sigma-Aldrich, St. Louis, MO, USA), β-actin (MAB1501, Millipore, Burlington, MA, USA), and Rb (554136, BD).
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