flag tagged itgb1  (Sino Biological)

Journal: Bone Research

Article Title: Collagen type II suppresses articular chondrocyte hypertrophy and osteoarthritis progression by promoting integrin β1−SMAD1 interaction

doi: 10.1038/s41413-019-0046-y

Figure Lengend Snippet: The ITGB1 receptor mediated the effect of COL2A1 on the BMP pathway and chondrocyte hypertrophy. a , b Immunoprecipitation with anti-BMPR1A ( a ) or anti-BMPR1B ( b ) antibody was performed in extracts of 293T cells treated with 100 μg·mL −1 COL2A1 or vehicle (0.05 mol·L −1 acetic acid) for 1 h, and then, immunoblotting was conducted with anti-phospho-serine/threonine and anti-BMPR1A/B antibodies. c − e Immunoblotting evaluation of p-ITGB1 and ITGB1 in chondrocytes of Col2a1 p.Gly1170Ser mutant mice ( c ), SW1353 and Hs819.T cells treated with COL2A1 or vehicle ( d ), and human articular chondrocytes transfected with COL2A1 siRNAs or negative control siRNA ( e ). f − h SW1353 and Hs819.T cells were pretreated with 10 μg·mL −1 ITGB1 blocking antibody or anti-human IgG antibody for 1 h and then treated with COL2A1 or vehicle for 1 h. Immunoblotting evaluation of p-ITGB1, p-SMAD1 S463/465 , ITGB1, and SMAD1 ( f ), and qPCR evaluation of ID1 , ID2 , DLX5 , RUNX2 , and COL10A1 ( g, h ) were performed. i Immunoblotting evaluation of SMAD1 in both nuclear (Nuc) and cytoplasmic (Cyto) extracts was performed in SW1353 and Hs819.T cells pretreated with ITGB1 blocking antibody or anti-human IgG and then treated with COL2A1 or vehicle. Data in ( g ) and ( h ) are presented as the mean ± SD ( n = 3). * P < 0.05. AC, articular chondrocyte; Blk ab, blocking antibody

Article Snippet: The pCMV3-FLAG-negative control vector plasmid (CV012), pCMV3-FLAG-SMAD1 plasmid (HG10715-CF), and pCMV3-HA-ITGB1 plasmid (HG10587-CF) were obtained from Sino Biological Inc.

Techniques: Immunoprecipitation, Western Blot, Mutagenesis, Transfection, Negative Control, Blocking Assay

Journal: Bone Research

Article Title: Collagen type II suppresses articular chondrocyte hypertrophy and osteoarthritis progression by promoting integrin β1−SMAD1 interaction

doi: 10.1038/s41413-019-0046-y

Figure Lengend Snippet: COL2A1 repressed BMP-SMAD1 signaling activation through facilitating the ITGB1−SMAD1 interaction and weakening the BMPR1A/B−SMAD1 interaction. a , b Immunoprecipitation was performed on 293T extracts with anti-SMAD1 antibody or anti-ITGB1 antibody, followed by immunoblotting with anti-SMAD1 antibody and anti-ITGB1 antibody. c 293T cells were transfected with SMAD1-Flag, ITGB1-HA or vector-Flag plasmids, and immunoprecipitation was carried out with anti-Flag antibody followed by immunoblotting with anti-HA antibody and anti-Flag antibody. d 293T cells were transfected with SMAD1-Flag, ITGB1-HA, or vector-HA plasmids, and immunoprecipitation was carried out with anti-HA antibody, followed by immunoblotting with anti-HA antibody and anti-Flag antibody. e SMAD1 protein was purified from 293T cell extracts expressing SMAD1-Flag and identified by Coomassie blue staining (left panel) and mass spectrometry (right panel). f Purified SMAD1-Flag protein was incubated with the product expressing ITGB1-HA, which had been linked to HA affinity agarose beads for 4 h at 4 °C, followed by bead washing and immunoblotting. g , h Immunoprecipitation was performed on 293T cells treated with 100 μg·mL −1 COL2A1 or vehicle (0.05 mol·L −1 acetic acid) for 1 h with anti-SMAD1 antibody ( g ) or anti-ITGB1 antibody ( h ), followed by immunoblotting with anti-SMAD1 antibody and anti-ITGB1 antibody. i , j Immunoprecipitation was performed on 293T cells treated with COL2A1 or vehicle with anti-SMAD1 antibody ( i ) or anti-BMPR1A antibody ( j ), followed by immunoblotting with anti-SMAD1 antibody and anti-BMPR1A antibody. k , l Immunoprecipitation was performed with anti-SMAD1 antibody ( k ) or anti-BMPR1B antibody ( l ) on 293T cells treated with COL2A1 or vehicle followed by immunoblotting with anti-SMAD1 antibody and anti-BMPR1B antibody. m , n 293T cells were pretreated with 10 μg·mL −1 ITGB1 blocking antibody or anti-human IgG antibody for 1 h and then treated with COL2A1 or vehicle for 1 h. Immunoprecipitation with anti-SMAD1 antibody ( m ) or anti-ITGB1 antibody ( n ) was conducted, followed by immunoblotting with anti-SMAD1 antibody and anti-ITGB1 antibody. Blk ab, blocking antibody

Article Snippet: The pCMV3-FLAG-negative control vector plasmid (CV012), pCMV3-FLAG-SMAD1 plasmid (HG10715-CF), and pCMV3-HA-ITGB1 plasmid (HG10587-CF) were obtained from Sino Biological Inc.

Techniques: Activation Assay, Immunoprecipitation, Western Blot, Transfection, Plasmid Preparation, Purification, Expressing, Staining, Mass Spectrometry, Incubation, Blocking Assay

Journal: Bone Research

Article Title: Collagen type II suppresses articular chondrocyte hypertrophy and osteoarthritis progression by promoting integrin β1−SMAD1 interaction

doi: 10.1038/s41413-019-0046-y

Figure Lengend Snippet: Functional mechanisms of COL2A1 on the BMP-SMAD1 pathway. The canonical BMP-SMAD1 pathway is initiated with the oligomerization of BMPR1 and BMPR2 induced by BMPs. BMPR2 phosphorylates BMPR1, and BMPR1, in turn, phosphorylates the C-terminus of SMAD1 (Ser463/465). C-terminal phosphorylated SMAD1 proteins partner with SMAD4 and translocate into the nucleus to initiate target gene transcription. ITGB1 is the major receptor for COL2A1, and upon its interaction with COL2A1, ITGB1 competes with BMPR1 for binding to SMAD1, thus inhibiting SMAD1 S463/465 phosphorylation and nuclear import. COL2A1 can also regulate SMAD1 activity via ITGB1-induced ERK1/2 phosphorylation. Activated ERK1/2 phosphorylates the linker region (Ser206) of both unphosphorylated and C-terminal phosphorylated SMAD1. The linker region-phosphorylated SMAD1 is resistant to phosphorylation by BMPR1 at the C-terminal, while the interaction between the C-terminal-phosphorylated SMAD1 and SMAD4 can also be interrupted by a second phosphorylation of the linker region of SMAD1. As a result, SMAD1 activation can be repressed via various signaling pathways specifically in response to COL2A1. BMP, bone morphogenetic protein

Article Snippet: The pCMV3-FLAG-negative control vector plasmid (CV012), pCMV3-FLAG-SMAD1 plasmid (HG10715-CF), and pCMV3-HA-ITGB1 plasmid (HG10587-CF) were obtained from Sino Biological Inc.

Techniques: Functional Assay, Binding Assay, Activity Assay, Activation Assay