The following antibodies were used in this study and were purchased from Santa Cruz Biotechnology unless otherwise noted (including dilutions/amounts used for immunofluorescence, Western blot [WB], and immunoprecipitation [IP]): ERα (HC-20 rabbit; 1:100 immunofluorescence, 1:200 WB; 3 µg IP), ERα (F-10 mouse; 3 µg IP), β1-integrin (LM534 mouse; 1:100 immunofluorescence; EMD Millipore), β1-integrin (M-106 rabbit; 1:300 WB; 3 µg IP), E-cadherin (H-108 rabbit; 1:1,000 WB), β-actin (C4 mouse; 1:10,000 WB), Rab11 (H-87 rabbit; 1:200 WB), Rab7 (sc-376362 mouse; 1:100 immunofluorescence), and caveolin 1 (sc-53564 mouse; 1:600 immunofluorescence; 1:200 WB). LAMP-1 (ab25630 mouse; 1:20 immunofluorescence), clathrin (ab2731 mouse; 1:500 immunofluorescence), and Lamin B1 (ab133741 rabbit; 1:243 immunofluorescence) were purchased from Abcam; and clathrin-HC (clone 23 mouse; 610500; 1:1,000 WB) was purchased from BD. HC-20 peptide was purchased from Santa Cruz Biotechnology. Secondary antibodies used for WB (1:5,000) were goat anti–mouse HRP-conjugated (AP308P) and goat anti–rabbit HRP conjugated (AP132P) purchased from EMD Millipore. Secondary antibodies used for immunofluorescence (1:500) were goat anti–mouse and goat anti–rabbit Alexa Fluor 488–, 555–, and 647–conjugated antibodies, all purchased from Thermo Fisher Scientific.
Integrin β1
Manufactured by Merck Group
Sourced in United States
Integrin β1 is a cell surface receptor protein that belongs to the integrin family. It plays a central role in cell-cell and cell-extracellular matrix interactions. Integrin β1 mediates the transmission of signals from the extracellular environment to the cell interior, and vice versa.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Merck Group (4 mentions)
α tubulin, by Merck Group (4 mentions)
Gapdh, by Cell Signaling Technology (3 mentions)
Vinculin, by Merck Group (3 mentions)
β catenin, by Merck Group (3 mentions)
Integrin α5, by Merck Group (3 mentions)
Erk1 2, by Cell Signaling Technology (3 mentions)
Fibronectin, by Abcam (2 mentions)
Collagen 3, by Abcam (2 mentions)
Total stat3, by Cell Signaling Technology (2 mentions)
Rock1, by Cell Signaling Technology (2 mentions)
Rock2, by Cell Signaling Technology (2 mentions)
P stat3, by Cell Signaling Technology (2 mentions)
Py397 fak, by Thermo Fisher Scientific (2 mentions)
P mlc2, by Cell Signaling Technology (2 mentions)
Tenascin c, by Abcam (2 mentions)
Gli 1, by Thermo Fisher Scientific (2 mentions)
Collagen 12a1, by Santa Cruz Biotechnology (2 mentions)
Mabt409, by Thermo Fisher Scientific (2 mentions)
Total fak, by BD (2 mentions)
28 protocols using integrin β1
1
Antibody Selection and Use Guidelines
2
Immunohistochemistry Antibody Panel
3
Protein-Protein Interaction Experiments Using Proximity Ligation Assay
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The protein–protein interaction experiments (growth factor receptors interactions with mucins and integrin complexes) were performed by using Duo Link II (Olink Bioscience, Watertown, MA, USA) Proximity Ligation Assay kit according to the manufacturer’s recommendation, as described previously [28 (link)]. Briefly, T3M4 WT and SC cells were grown on coverslips and then incubated with primary antibodies specific to MUC16 (AR9.6, Quest PharmaTech, Inc., AB, Canada), α4-integrin (Cell Signaling Technology, Danvers, MA, USA), and β1-integrin (EMD Millipore, Billerica, MA, USA), and then the primary antibodies were incubated with PLA probes anti-rabbit PLUS, anti-mouse MINUS. Experiments omitting the primary antibody used for single recognition PLA or either one of the primary antibodies used in double recognition PLA served as a negative control. The fluorescence images were captured under a Zeiss LSM 510 laser scanning confocal microscope (Carl Zeiss, Inc., Thornwood, NY, USA) at Confocal Laser Scanning Fluorescence Microscopy Core Facility, UNMC. The signal/dots per cell were quantified by using blob finder software.
4
Immunohistochemistry Antibody Panel
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Antibodies were as follows: a-SMA (Sigma-Aldrich C6198, 1:1000), GLi-1 (Thermo Scientific PA5-32206, 1:500), Tenascin C (Abcam AB108930, 1:500), Fibronectin (Abcam AB2413, 1:500), Collagen 12A1 (Santa Cruz Biotechnology E-15 sc-68449, 1:200), Sox2 (Abcam ab97959, 1:500), Vimentin (Cell Signaling 5741S, 1:200), FAP (Abcam AB53066, 1:500), Collagen III (Abcam AB7778, 1:500), YAP (Cell signaling 4912, 1:200), β1integrin (EMD Millipore MABT409, 1:500), pY397-FAK (Invitrogen 44625, 1:200), total FAK (BD Biosciences 610088, 1:1,000), ROCK1 (Cell Signaling C8F7, 1:1,000), ROCK2 (Cell Signaling D1B1, 1:1,000), p-MLC2 (Cell Signaling 3671, 1:200), p-MyPT1 (Millipore ABS45, 1:200), p-Stat3 (Cell Signaling 9145, 1:200), p-SMAD2/3 (Cell Signaling 8828, 1:200), total Stat3 (Cell Signaling 9132, 1:1,000), GAPDH (Cell Signaling 2118, 1:5,000), CD45 (BD Biosciences 550539, 1:200), CD68 (Thermo Scientific Ab-3, 1:200), Alexa Fluor-conjugated goat secondary anti–mouse IgG and anti–rabbit IgG antibodies (Invitrogen A11012, 1:1,000 and A11005, 1:1,000) and HRP-conjugated rabbit secondary antibody (GE health care life sciences NA934VS, 1:5,000).
5
Comprehensive Protein Expression Analysis
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Western blotting and indirect immunofluorescence were performed using the following primary antibodies: β actin (A5441, Sigma-Aldrich, Ireland); β1 integrin (#1952, Merck Millipore, Ireland); cyclin B1 (#4135), estrogen receptor α (#8644), HER2 (#4290), EGFR (#4267), IGF-1R (#3027), pan AKT (#2920), pan ERK (#4696), pan p-AKT (#4060), pan p-ERK (#4377), p-EGFR (#3777), progesterone receptor A/B (#8757) [all from Cell Signaling Technology Europe, Leiden, Netherlands]; cyclin D1 (ab16663) and N-cadherin (ab19348) [both from Abcam, Cambridge, UK]; E-cadherin (#610181, BD Biosciences, San Jose, CA, USA); GAPDH (sc47724) and vimentin (sc32322) [both from Santa Cruz Biotechnology, Dallas, TX, USA]. Secondary antibodies for western blots were IRDye 800CW or 680RD (LI-COR Biosciences UK Ltd., Cambridge, UK); and the secondary antibody used for β actin indirect immunofluorescence was DyLight 488 donkey anti-mouse (Jackson ImmunoResearch Europe Ltd., Ely, UK).
6
Kidney Protein Analysis by Immunoblotting
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Equal amounts of protein from four or more kidney lysates per experimental condition were pooled and resolved by SDS-PAGE. Using a standard technique, immunoblotting was performed with the following primary antibodies: WT1, podocin, nephrin, laminin, sema3a (sc-28867; Santa Cruz), β3-integrin (sc-14009; Santa Cruz), β1-integrin (AB1952; EMD Millipore), neuropilin1 (17 (link)), matrix metalloproteinase (MMP)-2 (MAB13434; EMD Millipore), MMP-9 (AB19016; EMD Millipore), plexinA1 (sc-25639; Santa Cruz), VEGF receptor 2 (2479; Cell Signaling Technologies), and MICAL1 (14818–1-AP; Proteintech). Actin (A2066; Sigma) or tubulin (Sigma) was used as a loading control. Signals were detected with appropriate horseradish peroxidase–conjugated secondary antibodies, visualized by chemiluminescence, and quantified using ImageJ software.
7
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 ).
8
Western Blot Antibody Validation
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Caspase-3 (9662), AKT (9272), p-AKT Ser-473 (9271), S6 (2217), p-S6 Ser-235/236 (4858), p-GSK3β Ser-9 (9336), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 14C10) antibodies were purchased from Cell Signaling Technology (Danvers, MA). AR (N-20), p-AR Ser-308 (sc-26406-R), and p21 (C-19) were from Santa Cruz Biotechnology (Dallas, TX). CDK5 antibody (DC34) was from Invitrogen (Carlsbad, CA), Hsc70 (SPA815) from StressGen (Victoria, Canada), GFP (JL-8) from Clontech (Mountain View), β-catenin from BD Biosciences (San Jose, CA), β1-integrin from Millipore (Billerica, MA), and PARP-1 (C-2-10) and actin (AC-40) from Sigma-Aldrich (St. Louis, MO). Secondary horseradish peroxidase (HRP) antibodies were from Promega (anti-rabbit; Fitchburg, WI) and GE Healthcare (anti-rat, anti-mouse; Cleveland, OH). Fluorescent secondary antibodies (Alexa Fluor 488 or 546) and Alexa Fluor 633–phalloidin were from Invitrogen.
9
Spinal Cord Immunohistochemistry Protocol
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Animals were killed using an overdose of halothane anesthesia and transcardially perfused with 4% paraformaldehyde in PBS. The spinal cords were dissected and fixed for 2 h with 4% paraformaldehyde and subsequently overnight in 30% sucrose in PBS. The spinal cords were then frozen in Tissue-Tek embedding compound and sectioned on a Leica (Deerfield, IL) CM3050S cryostat.
20 µm thick frozen sections were cut on a leica (CM3050S) Cryostat and collected on Superfrost Bond Rite slides (Richard Allen Scientific). Every fifth section was placed on the same slide such that each adjacent section was 80 µm away from its neighbor. Four sections were placed on each slide and hence the width of the frozen cord spanned on each slide was 240 µm. We roughly averaged about 20 slides per cord. Sections were processed for immunohistochemistry in the same manner as described for the cells. Primary antibodies used were: GFAP (Sigma, mouse IgG1 1∶500), β1integrin (Millipore, rat IgG, 1∶500), HUTS-4 (Millipore, mouse IgG1 1∶500).
20 µm thick frozen sections were cut on a leica (CM3050S) Cryostat and collected on Superfrost Bond Rite slides (Richard Allen Scientific). Every fifth section was placed on the same slide such that each adjacent section was 80 µm away from its neighbor. Four sections were placed on each slide and hence the width of the frozen cord spanned on each slide was 240 µm. We roughly averaged about 20 slides per cord. Sections were processed for immunohistochemistry in the same manner as described for the cells. Primary antibodies used were: GFAP (Sigma, mouse IgG1 1∶500), β1integrin (Millipore, rat IgG, 1∶500), HUTS-4 (Millipore, mouse IgG1 1∶500).
10
Protein Extraction and Western Blotting
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For protein extraction, cells were lysed in T-Per Tissue Protein Extraction Reagent (Thermo), 1 mM PMSF (Sigma), 1× Halt Phosphatase Inhibitor Cocktail (Thermo), and Complete Protease Inhibitor Tablet (Roche). Total protein extract was resolved by SDS-PAGE on precast gels (Bio-Rad) and then transferred to Immuno-blot PVDF Membranes (Bio-Rad) using a Bio-Rad mini-Protein II Transfer system. Membranes were rinsed in 0.1% Tween (Sigma)/TBS (20 mM Tris-HCl pH 7.5, 150 mM NaCl), blocked for 1 h at RT in 5% low-fat milk (Carnation)/0.1% Tween/TBS (blocking buffer), then incubated with primary antibodies in blocking buffer overnight at 4 °C. Primary antibodies against following antigens were used at specified dilutions: β1-integrin, 1:1000 (Millipore); Cyclin A, 1:200; Cyclin B1, 1:200; Cyclin D1, 1:200; Cyclin D2, 1:200; Cyclin D3, 1:200; Cyclin E, 1:200; Gapdh, 1:5000; pErk, Erk, pAkt, Akt, FAK, and pFAK, all at 1:1000. After washing in 0.1% Tween/TBS, appropriate secondary antibodies (Amersham, Invitrogen, and Bio-rad) were diluted 1:10,000 in blocking buffer and incubated for 1 h at RT.
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