Protein extracts were prepared by lysing germ cells isolated as describe above (“RNA-seq” section) or K562 cells in RIPA Lysis buffer (Millipore) containing 50 mM Tris–HCl, pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 500 mM NaCl, 1 mM EDTA, and 1× protease inhibitor cocktail (Roche Applied Science). Protein samples were resolved by SDS–polyacrylamide gel electrophoresis, transferred to a polyvinylidene difluoride membrane, and incubated with the indicated antibodies. Detections were performed using ECL reagents (Pierce). Kit protein was detected with 1:2000 anti-c-kit (R&D Systems AF1356) and 1:2000 of anti-goat secondary antibody (Santa Cruz sc2020). CTCF protein was detected with anti-CTCF B-5 (Santa Cruz sc-271514) and 1:2000 of an anti-mouse secondary antibody (Santa Cruz sc-2005). Tubulin was detected using 1:5000 anti-α Tubulin B-7 (Santa Cruz sc-5286) and 1:2000 of an anti-mouse secondary antibody (Santa Cruz sc-2005). Images were quantified using the ImageJ (v1.48) software and normalized to Tubulin.
Anti α tubulin b 7
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Anti-α-tubulin (B-7) is a mouse monoclonal antibody that specifically recognizes the α-tubulin protein. This antibody can be used for the detection and analysis of α-tubulin in various applications, such as Western blotting, immunohistochemistry, and immunocytochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Anti goat secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Anti c kit, by R&D Systems (1 mentions)
Ripa lysis buffer, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Typhoon 9400 variable mode imager, by GE Healthcare (1 mentions)
Anti 14 3 3ζ antibody, by Santa Cruz Biotechnology (1 mentions)
Precast gel, by Bio-Rad (1 mentions)
Semi dry transfer system, by Bio-Rad (1 mentions)
Pvdf membrane, by GE Healthcare (1 mentions)
Recombinant human icam 1 fc, by R&D Systems (1 mentions)
Sb203580, by Cell Signaling Technology (1 mentions)
Tnf α, by R&D Systems (1 mentions)
Anti mouse secondary, by Santa Cruz Biotechnology (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Bradford protein assay, by Bio-Rad (1 mentions)
Anti cyclin d3 dcs22, by Cell Signaling Technology (1 mentions)
Palbociclib, by LC Laboratories (1 mentions)
Ribociclib, by LC Laboratories (1 mentions)
Anti phospho syk, by Cell Signaling Technology (1 mentions)
Anti syk, by Cell Signaling Technology (1 mentions)
9 protocols using anti α tubulin b 7
1
Protein Extraction and Western Blot Analysis
2
Immunoblotting of Platelet TLR4 Activation
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The isolated human platelets were treated with either modified Tyrode’s-HEPES buffer (resting control) or 0.5 μg/mL CRP-XL for five minutes in an aggregometer prior to lysis in reducing sample treatment buffer [RSTB; 69 mM sodium dodecyl sulphate, 5% (v/v) 2-mercaptoethanol, 10% (v/v) glycerol, and 25 mM Tris-HCl]. Subsequently, the platelet lysates were boiled at 90 °C for 10 minutes and analysed by SDS-PAGE in 4–15% pre-cast gels (Bio-Rad, UK) and then transferred to a PVDF membrane (GE Healthcare, UK) using a Semi-Dry Transfer System (Bio-Rad, UK). The membrane was blocked with 5% (w/v) bovine serum albumin (BSA) in PBS with 0.1% (v/v) Tween-20 (PBS-T) for 1 hour at room temperature. Then it was incubated overnight at 4 °C with anti-TLR4 antibody (1/250 dilution) and for 1 hour at room temperature with anti-α-tubulin [B-7] or anti-14-3-3ζ antibody (1/2000 dilution) (Santa Cruz Biotechnology, USA). 30 μg of HEK-293 or U251-NF-κB-GFP-Luc cell lysates were used as a TLR4-negative or -positive control respectively. The primary antibodies were detected by using Cy5-conjugated goat anti-mouse IgG (1/2500 dilution) (Thermo Fisher Scientific, UK) in a Typhoon 9400 variable mode imager (GE Healthcare, UK) (488 V) and images were analysed using ImageJ.
3
Multivalent Polymer-Mediated Cell Adhesion
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The multivalent polymers PAA [HOCH2(HOCH)4CH2NH-PAA] and Ley-PAA, and the multivalent biotinylated polymers PAA-biotin and Ley-PAA-biotin were purchased from GlycoTech Corporation (Gaithersburg, MD, USA). Recombinant human ICAM-1-Fc and TNF-α were purchased from R&D Systems (Minneapolis, MN, USA). The p38 MAPK inhibitor SB203580 and anti-phosphor-p38 MAPK (Thr180/Tyr182) (D3F9) mAb were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-p38 MAPK polyclonal Ab (C-20), anti-TM (D-3), anti-L-selectin (lam1-116) and anti-α-tubulin (B-7) mAbs were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-TS1/18 mAb and Alexa Fluor 488- and 546-conjugated secondary antibodies were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Anti-MOMA-2 mAb was purchased from Abcam (Cambridge, UK).
4
Western Blot Analysis of MTAP Protein
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Log-phase CEM and Molt4 cells were harvested by centrifugation after culture in RPMI 1640 supplemented with 10 % FBS. After brief centrifugation, cell pellets were lysed in RIPA buffer containing a commercial protease inhibitor mix (Roche, Nutley, NJ, USA). After protein quantification by the Bradford protein assay (Bio-Rad Laboratories, Hercules, CA, USA), proteins were resolved by 10 % SDS-PAGE and transferred onto a nitrocellulose membrane (Bio-Rad Laboratories). After blocking the membrane with 5 % nonfat dry milk prepared in Tris-buffered saline +0.1 % Tween-20, the membrane was incubated with the desired primary antibody according to the manufacturer’s directions at 4 °C overnight. The membrane was washed in Tris-buffered saline +0.1 % Tween-20 and incubated for 2 h at room temperature with the appropriate peroxidase-conjugated secondary antibody. Bands were visualized using an enhanced chemiluminescence kit (Pierce, Thermo Fisher Scientific, Rockford, IL, USA). Anti-MTAP (42-T), anti-α-tubulin (B-7) and anti-mouse secondary were purchased from Santa Cruz Biotechnologies (Dallas, TX, USA).
5
Comprehensive Cell Cycle Protein Analysis
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The following antibodies: anti-GAPDH (FL-335), anti-K19 (A53-B/A2), anti-K8 (C51), anti-K18 (C-04), anti-cyclin D1 (DCS-6), anti-cyclin E (E-4), anti-cyclin A (H-3), anti-CDK1 (CDC2 p34(17)), anti-CDK4 (DCS-35), anti-CDK7 (C-4), anti-E2F1 (KH95) and anti-α tubulin (B-7) were from Santa Cruz Biotechnology (Santa Cruz, CA); anti-Rb (4H1), anti-phospho-Rb (Ser807/811), anti-E2F1, anti-cyclin D3 (DCS22) and anti-cyclin B1 (D5C10) were from Cell Signaling Technology (Danvers, MA). Palbociclib and ribociclib were from LC Laboratories (Woburn, MA) and THZ1 was from Cayman chemical (Ann Arbor, MI).
6
Antibody Panel for Macrophage Signaling
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Anti-MGL1 antibody LOM-8.7 was previously generated in our laboratory (Kimura et al. 1995 (link)). The following antibodies were purchased: anti-Phospho-Syk (Tyr525/526, Cell Signaling Technology, Danvers, MA, USA), anti-Syk (Cell Signaling Technology), fluorescein isothiocyanate-conjugated anti-CD11b (Biolegend, San Diego, CA, USA), phycoerythrin-conjugated anti-F4/80 (eBioscience, San Diego, CA, USA). The following antibodies were a kind gift from Profs. Ichijo Hidenori (The University of Tokyo, Tokyo, Japan) and Atsushi Matsuzawa (Tohoku University, Sendai, Japan), anti-Phospho-p38 MAPK (Thr180/Tyr182, Cell Signaling Technology), anti-IκB-α (H-4, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-αTubulin (B-7, Santa Cruz Biotechnology), anti-Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204, clone E10, Cell Signaling Technology), anti-ERK 2 (C-14, Santa Cruz Biotechnology).
7
SDS-PAGE Immunoblotting Reagents
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Reagents for SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting were from Bio-Rad Laboratories (Hercules, CA). Tris, aprotinin, adenosine triphosphate (ATP), dithiothreitol, phenylmethylsulfonyl fluoride, Triton X-100, Tween 20, glycerol, and bovine serum albumin (BSA) (fraction V) were from Sigma Chemical Co. (St. Louis, MO). Nitrocellulose paper (BA85, 0.2 mm) was from Schleicher & Schuell (Keene, NH). Ketamine hydrochloride was from Cristália (Itapira SP, Brazil). The chemiluminescent kit was from Thermo Fisher Scientific (Rockford, IL, USA). The following antibodies were used: Anti-IL6 (M-19) and anti–α-tubulin (B-7) were from Santa Cruz Biotechnology; anti-pERK1/2 (Thr202/Tyr204), anti-p44/42 MAPK (ERK1/2), anti-pACC (Ser79), anti-ACC, anti–pAMPK (Thr172), anti-AMPKα, and anti–α-tubulin were from Cell Signaling Technology. Secondary antibodies were from Thermo Fisher Scientific. Recombinant IL6 was from Calbiochem (San Diego, CA, USA). PD98059 was from LC Laboratory (Woburn, MA). Unless otherwise specified, routine reagents were purchased from Sigma Chemical Co. (St. Louis, MO). The doses administered in each experimental group are given below.
8
Cellular Signaling Pathway Analysis
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Chemical reagents included bovine serum albumin (BSA) (Sigma-Aldrich, B2518), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich, M5655), trypan blue (Sigma-Aldrich, T6146), propidium iodide (PI) (Sigma-Aldrich, P4864), GSKJ4 (Sigma-Aldrich, SML0101), PD98059 (Sigma-Aldrich, P215), PKF118-310 (Sigma-Aldrich, K4394), MG132 (Alexis 133407-82-6), and H89 (Sigma-Aldrich, #B1427). Antibodies obtained from Santa Cruz Biotechnology: anti-nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p65(A) (sc-109), anti-Ub (P4D1) (sc-8017), anti-α-tubulin (B-7) (sc-5286). Antibodies purchased from Cell Signaling Technology: anti-CREB (#9198S), anti-p44/42 mitogen-activated protein kinase (MAPK) (ERK1/2) (#9102), anti-p-CREB (Ser133, #9198), anti-phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (#9101). Anti-vinculin (ab13007) and anti-H4 (ab10158) were bought from Abcam. Other antibodies used were anti-β-actin AC-74 (Sigma-Aldrich, A2228) and anti-H3K27me3 (Diagenode, C15410195). Conjugate horseradish peroxidase (HRP) goat anti-rabbit (GtxRb-003-DHRPX) and goat anti-mouse (GtxMu-003-EHRPX.0.05) (Immunoreagents Inc.) were employed for immunoblotting detection.
9
Molecular Pathway Regulation in Cell Lines
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SFN-Cys and SFN-NAC were purchased from Santa Cruz Biotechnology (CA, USA). LipofectamineTM RNAiMAX was purchased from Invitrogen-Life Technologies (CA, USA). Anti-ERK1/2 (1:1000), anti-phospho-ERK1/2 (1:1000), and phosphorylated ERK1/2 inhibitor PD98059 were purchased from Cell Signaling Technology, Inc. (Shanghai, China). Anti-Claudin-1 (1:2000) was purchased from Abcam (MA, USA), anti-Claudin-5 (1:1000) was purchased from Santa Cruz (CA, USA), and anti-Claudin-7 (1:100) was purchased from Sangon Biotech, Ltd (Shanghai, China). Anti-LC3 was purchased from Cell Signaling Technology (CO, USA). Anti-Tau (Tau46) and anti-α-tubulin (B-7) were purchased from Santa Cruz (TX, USA). Autophagy inhibitor 3-Methyladenine (3-MA) was bought from Sigma (Hongkong, China) and Bafilomycin A1 (Baf-1) was from Selleck (Shanghai, China).
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