SNU449 cells were transiently transfected with strep-tagged TM4SF5 11 (link) (or mock-transfected) for 24 h and then treated with Ab27 or Ab79 (10 µg/ml) for an additional 24 h. Cells were lysed in lysis buffer (20 mM HEPES, pH 7.4, 150 mM NaCl, 2 mM MgCl2, 2 mM CaCl2) containing 1% Brij58. Whole cell lysates (850 µg/condition) were immunoprecipitated with streptavidin beads (Thermo Fisher Scientific, Waltham, MA, USA) at 4ºC for 16 h. The protein complexes were washed twice with ice-cold lysis buffer and then twice with PBS. The immunoprecipitated proteins were eluted by boiling in SDS sample buffer and analyzed by immunoblotting with anti-Strep tag (IBA; Olivette, MO, USA), anti-integrin α2 (Chemicon, Temecula, CA, USA), anti-integrin α5 (BD Biosciences), and anti-CD44 (BioLegend; San Diego, CA, USA) antibodies.
Anti integrin α2
Manufactured by Merck Group
Sourced in United States
Anti-integrin α2 is a lab equipment product that targets the integrin α2 subunit. It is used in research applications to study the role of integrin α2 in various biological processes.
Automatically generated - may contain errors
Lab products found in correlation
Anti integrin α5, by BD (2 mentions)
Anti aqp4, by Merck Group (2 mentions)
Anti sma, by Merck Group (2 mentions)
Anti actin, by Merck Group (2 mentions)
Anti sm22α, by GeneTex (2 mentions)
Anti myocardin, by R&D Systems (2 mentions)
Mab4028, by Merck Group (2 mentions)
Anti occludin, by Thermo Fisher Scientific (2 mentions)
Anti claudin 5, by Thermo Fisher Scientific (2 mentions)
Anti pdgfrβ, by Cell Signaling Technology (2 mentions)
Anti zo 1, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Protein assay kit, by Bio-Rad (2 mentions)
Hrp conjugated secondary antibody, by Jackson ImmunoResearch (2 mentions)
Streptavidin beads, by Thermo Fisher Scientific (1 mentions)
Anti cd44, by BioLegend (1 mentions)
Anti integrin α4, by Santa Cruz Biotechnology (1 mentions)
Anti sox2, by Cell Signaling Technology (1 mentions)
Anti bmi 1, by Merck Group (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
6 protocols using anti integrin α2
1
Immunoprecipitation of TM4SF5 Complexes
2
Whole-cell Lysates Preparation and Immunoblotting
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Whole-cell lysates were prepared using RIPA buffer as described previously [26 (link)] and analyzed using the following primary antibodies: anti-integrin α4, anti-SP1, and anti-GAPDH (Santa Cruz Biotechnology, Dallas, TX); anti-integrin α2 (Chemicon International, Temecula, CA); anti-myc (Upstate Biotechnology, Lake Placid, NY); anti-cyclin D2, anti-phospho-c-Jun(S63), anti-c-Jun, anti-phospho-AKT, anti-AKT, anti-survivin, anti-Bcl-2, anti-SLUG, anti-SOX2, anti-CD133, anti-ABCB1, and anti-KLF4 (Cell Signaling Technology, Danvers, MA); anti-BMI1 (Millipore, Temecula, CA), anti-integrin α5 and anti-E-cadherin (BD Biosciences, San Jose, CA); anti-vimentin and anti-flag (Sigma); anti-TWIST1 (Abcam, Cambridge, MA); and anti-TMPRSS4 (in-house) [26 (link)].
3
Quantitative Analysis of Protein Markers
4
Quantitative Analysis of Protein Markers
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Total protein concentration from the lysates was determined using the Bio-Rad protein assay kit. Equal amount of protein was loaded and separated in 10% or 12% SDS-PAGE, followed by transfer to PVDF membrane (Millipore). The membranes were then incubated with anti-AQP4 (Millipore, AB3594, 1:500), anti-occludin (Invitrogen, 71-1500, 1:1000), anti-ZO-1 (Invitrogen, 40-2200, 1:1000), anti-Claudin-5 (Invitrogen, 34-1600, 1:1000), anti-PDGFRβ (Cell Signaling, 3169S, 1:500), anti-SMA (Sigma, F3777, 1:1000), anti-SM22-α (GeneTex, GTX113561, 1:1000), anti-integrin α2 (Millipore, AB1936, 1:500), anti-myocardin (R&D, MAB4028, 1:500) and anti-actin (Sigma, A5441, 1:2000) antibodies at 4 overnight, followed by incubation with HRP-conjugated secondary antibodies (Jackson ImmunoResearch Lab). The proteins were visualized by SuperSignal West Pico Chemiluminescent Substrate (Pierce). The density of bands was normalized to actin and quantified using NIH Image J.
5
Integrin-Mediated Cell Adhesion Enhancement
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The involvement of integrins in the enhancement of cell adhesion by the ROCK inhibitor was evaluated by seeding MCECs (5 × 103 cells/well) in 96-well plates in the presence or absence of integrin-neutralizing antibodies (2 μg/mL): anti-α1 integrin (Merck Millipore, Billerica, MA), anti-α2 integrin (Merck Millipore, Billerica, MA), anti-α3 integrin (Merck Millipore, Billerica, MA), anti-α4 integrin (Merck Millipore, Billerica, MA), anti-α5 integrin (Merck Millipore, Billerica, MA), anti-α6 integrin (Merck Millipore, Billerica, MA), anti-αV integrin (Merck Millipore, Billerica, MA), anti-α6 integrin (Merck Millipore), and anti-β1 integrin (R&D systems Inc., Minneapolis, MN). The effect of inhibiting phosphorylation of MLC and RhoA activity on cell adhesion was also evaluated by seeding MCECs with blebbistatin (10 μM) and C3 (300 ng/ml), respectively. Three hours after seeding, the numbers of adherent cells were determined with the CellTiter-GloTM luminescent cell viability assay (Promega Corporation, Madison, WI) according to the manufacturer’s instructions. The number of adhered cells was determined using a VeritasTM microplate luminometer (Promega Corporation).
6
FACS Analysis of Stem Cell Markers
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FACS analysis was performed as described previously [13 (link)]. The following primary antibodies were used: anti-CD44v9 (Cosmo Bio Co., Ltd., Japan), anti-CD24 (Santa Cruz Biotechnology, Dallas, TX, USA), anti-α2-integrin (Merck Millipore, Billerica, MA, USA), and anti-β1-integrin (Abcam, Cambridge, UK). Mean fluorescence intensity (MFI) was calculated by subtracting the intensities of the control samples. These experiments were independently performed three times.
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