Antibodies for eIF4G1 (Cell Signaling #2858); Cyclin D1 (Santa Cruz sc-20044); Rb (Cell Signaling #9309); p-Rb (Cell Signaling #9308); β-Tubulin (Developmental Studies Hybridoma Bank E7); β-Actin (Sigma A2228) were used to probe respective proteins of interest. Inhibitor for eIF4G-eIF4E complex (4EGI-1), (Catalog No. S7369) were purchased from Selleck Chemicals.
β tubulin
Manufactured by Developmental Studies Hybridoma Bank
Sourced in United States
β-tubulin is a core component of microtubules, which are essential cytoskeletal structures involved in various cellular processes. It plays a crucial role in the formation and stability of the mitotic spindle during cell division.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Merck Group (2 mentions)
Flag tag (flag), by Merck Group (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
P jnk, by Cell Signaling Technology (2 mentions)
Cyclin d1, by Santa Cruz Biotechnology (1 mentions)
Cul4a, by Cell Signaling Technology (1 mentions)
Flag m2, by Merck Group (1 mentions)
Ni nta agarose resin, by Qiagen (1 mentions)
H3k27ac, by Cell Signaling Technology (1 mentions)
Dulbecco modified eagle medium (dmem), by Avantor (1 mentions)
Uea 1, by Vector Laboratories (1 mentions)
Bclaf1, by Santa Cruz Biotechnology (1 mentions)
Hnrnpu, by Thermo Fisher Scientific (1 mentions)
Aal biotin, by Vector Laboratories (1 mentions)
Anti mouse irdye 680rd, by LI COR (1 mentions)
Plasmotest kit, by InvivoGen (1 mentions)
M iggκ bp hrp, by Santa Cruz Biotechnology (1 mentions)
Vimentin, by Abcam (1 mentions)
Taxol, by Merck Group (1 mentions)
Glu tubulin, by Abcam (1 mentions)
17 protocols using β tubulin
1
Analyzing Signaling Protein Regulation
2
Antibody Generation and Protein Purification Protocol
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BRWD2/PHIP antibodies (Shilatifard, nos. 833 and 834) were raised against a protein fragment spanning BRWD2/PHIP amino acids 1681–1821 (sequence ID NP_060404.4). Drosophila dBRWD3 antibody (Shilatifard, no. 385) was raised against amino acids 976–1495 (sequence ID NP_732982.1). Proteins were expressed from the pET16 plasmid in Rosetta2 E. coli cells and purified using Ni-NTA agarose resin (Qiagen). Rabbit immunization was performed by Pocono Rabbit Farm and Laboratory. Other antibodies used in this study were H3K4me1 (Shilatifard Laboratory, no. 24), H3K4me2 (Shilatifard Laboratory, no. 27), H3K4me3 (Shilatifard Laboratory, no. 680), H3K27ac (Cell Signaling Technology, no. 8137), H2A.Z (Cell Signaling Technology, no. 2718), H3K27me3 (Shilatifard Laboratory, no. 67), DDB1 (Cell Signaling Technology, no. 6998), CUL4A (Cell Signaling Technology, no. 2699), CHD4 (Cell Signaling Technology #12011), PHF6 (Santa Cruz Biotechnology, no. sc-365237), β-Tubulin (E7, Developmental Studies Hybridoma Bank), Histone H3 (Shilatifard Laboratory, no. 42), and Flag-M2 (Sigma, no. F1804).
3
Cell Line Authentication and Mycoplasma Detection
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All cell lines used in this manuscript were validated for identity by the H. Lee Moffitt Cancer Center Molecular Genomics Core within 2 years of testing and were confirmed to be mycoplasma-free using the Plasmo Test kit (InvivoGen, San Diego, CA, USA). Cell lines were cultured in DMEM (VWR, Radnor, PA, USA) supplemented with heat inactivated fetal bovine serum 1:10 (Peak Serum, Denver, CO, USA). Antibodies: RPS3 (Bethyl, Montgomery, TX, USA), HNRNPU (ThermoFisher, Waltham, MA, USA), BCLAF1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), EIF3K (Bethyl), FLAG (Sigma, St. Louis, MO, USA), β-actin (Sigma), β-tubulin (Developmental Studies Hybridoma Bank, DSHB, Iowa City, IA, USA), LaminA/C (DSHB). UEAI, AAL, UEAI-488, AAL-488, UEAI-biotin, AAL-biotin were purchased from Vector Laboratories (Burlingame, CA, USA). Secondary antibodies: anti-rabbit-HRP (Santa Cruz Biotechnology, Inc.), m-IgGκ-BP-HRP (Santa Cruz Biotechnology, Inc.), streptavidin-800 (Life Technologies, Carlsbad, CA, USA), IRDye 680RD anti-mouse (LI-COR, Lincoln, NE, USA).
4
Immunostaining of Cytoskeletal Proteins
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Cells were fixed in 4% paraformaldehyde at room temperature for 15 min or 100% methanol at –20°C for 10 min and processed for immunostaining using standard procedure. Primary antibodies used were against Rudhira (Jain et al., 2012 (link)), Vinculin, α-tubulin, Ac-tubulin (Sigma Chemical Co.), β-tubulin (Developmental Studies Hybridoma Bank [DSHB]; ThermoFisher Scientific; Abcam), Paxillin, FAK, β1 Integrin (Merck), vimentin, Glu-tubulin, EB1 (Abcam), plectin (Santa Cruz Biotechnology), GFP (ThermoFisher Scientific), pFAK, and pY (Cell Signaling Technologies). Secondary antibodies were coupled to Alexa-Fluor 488 or Alexa-Fluor 568 or Alexa-Fluor 633 (Molecular Probes). Phalloidin was conjugated to Alexa-Fluor 633 (Molecular Probes). Nocodazole, ROCK inhibitor (ROCKi, Y27632), and Taxol (paclitaxel) were from Sigma Chemical. Cells were treated with ROCKi or Taxol for 1 h and processed for immunostaining with Paxillin, tubulin or vimentin antibodies, or Phalloidin, as indicated (Figure 5, A–E ).
5
Protein Expression Analysis by Western Blot
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Whole-cell extracts were harvested using radioimmunoprecipitation (RIPA) buffer supplemented with protease and phosphatase inhibitors (Roche), run on SDS–PAGE gels (Life Technologies), and then transferred onto nitrocellulose membranes (0.45 µm, BioRad). Immunoblotting was carried out using antibodies against the following proteins in 5% bovine serum albumin in TBST at 4 °C overnight: GLI1 (Cell Signaling, 2534), β-tubulin (Developmental Studies Hybridoma Bank, E7), phospho-Smad2/3 (Abcam ab52903), Smad2 (BD Biosciences 610842), phospho-p38 MAPK (Cell Signaling, 4511), phospho-Smad1/5/8 (Cell Signaling, 9511). Then membranes were washed and incubated with secondary antibodies IRDye 680LT donkey anti-mouse IgG and IRDye 800CW IgG donkey anti-rabbit IgG (LI-COR). All blots were imaged using the LI-COR Odyssey image system and ImageStudioLite software.
6
Western Blot Analysis of β-Catenin
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Cellular protein from cultured cells and xenograft tumors were homogenized in RIPA lysis buffer with a mixture of protease (Roche) and phosphatase (Millipore Sigma) inhibitors. Protein concentration was determined using the Bradford assay (Bio-Rad), and samples were run on a 7% PAGE gel. Primary antibodies used were mouse antinonphosphorylated (Ser37/Thr41) βcatenin (8E&, 1:500; MilliporeSigma), mouse anti-β-actin (1:10,000; MilliporeSigma), and β-Tubulin (E7, University of Iowa Developmental Studies Hybridoma Bank). Membranes were probed with horseradish peroxidase-linked donkey anti-mouse IgG (1:40,000; Millipore Sigma), and detection of protein was conducted using ECL Western blotting detection reagent (Thermo Fisher). All Western blots are representative images from a minimum of three biological replicates.
7
Caspase-1 Activation in Bacteria Infection
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BMMs were stained with FAM-YVAD-fmk (caspase-1 FLICA; Immunochemistry Technologies, Bloomington, MN) beginning 5 min before infection with S. typhimurium or F. novicida at an MOI of 30. Cells were fixed at the indicated times with 4% paraformaldehyde for 20 min at 37°C. Blocking and permeabilization were carried out for 30 min with 0.1% Triton X-100 and 5% normal donkey serum in PBS. Primary antibody staining was carried out against β-tubulin (1:100; Developmental Studies Hybridoma Bank, Iowa City, IA; E7) or α-tubulin (1:100; Cell Signaling Technologies). Secondary antibodies conjugated to Alexa Fluor 555 and Alexa Fluor 647 (Life Technologies) were used for staining. Imaging was done with a Nikon TE2000 inverted fluorescence microscope and an EVOS fluorescence microscope.
8
Immunofluorescence Assay for Cellular Markers
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Antibodies included β-tubulin (1:250, E7; Developmental Studies Hybridoma Bank), PKC ζ (1:100, sc-216; Santa Cruz Biotechnology); centrosomin (1:500; Rusan Lab); phosphohistone H3 Ser10 (1:1,000, 06-570; EMD Millipore); FLAG (1:500, F1804; Sigma-Aldrich); and Deadpan (1:100, ab195172; Abcam). Secondary antibodies conjugated to Alexa Fluor 488, 568, and 647 were obtained from Life Technologies and used at 1:500.
9
Western Blot Analysis of Synaptic Proteins
10
Antibody Validation for Western Blot and Immunofluorescence
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GFP-TFEB, MitoTimer, mCherry-Parkin and blasticidin-resistance expression plasmids were from Addgene plasmid repository. Antibodies and their respective dilutions used in immunoblotting (IB) and immunofluorescence (IF) were: AKT (C67E7) (Cell Signaling Technology (CST), IB: 1:1000); β-actin (Abcam, IB: 1:10,000); β-tubulin (Developmental Studies Hybridoma Bank (DSHB), IB: 1:2000); ERK (clone 16) (BD Bioscience, IB: 1:1000); GFP (Roche, IB: 1:3000); LAMP1 (H4A3) (DSHB, IB: 1:1000, IF: 1:300); LC3 (CST for IB, 1:1000; Novus for IF, 1:300); mTOR (CST, IB: 1:1000); p70/S6K (CST, IB: 1:1000); PINK1 (Abcam, IF: 1:500); phospho-AKTT308 (CST, IB: 1:1000); phospho-AKTS473 (CST, IB: 1:1000); phospho-mTORS2448 (CST, IB: 1:1000); phospho-p44/42 MAPK (ERK1/2)T202/Y204 (CST, IB: 1:1000); phospho-p70/S6KT389 (CST, IB: 1:1000); phospho-TFEBS142 (Millipore, IB: 1:3000); TOMM20 (Abcam, IB: 1:1000, IF: 1:1000). For IB, mouse and rabbit horseradish peroxidase (HRP) conjugated secondary antibodies (Sigma) were used at 1:10,000 dilution. For IF, Alexa Fluor-conjugated secondary antibodies (Invitrogen) were used at 1:800 dilution.
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