Journal: International journal of molecular sciences

Article Title: Loss of Tyrosine Phosphatase Mu Promotes Scoliosis Progression Through Osteopontin-α5β1 Integrin Signaling and PIPK1γ90 Activity.

doi: 10.3390/ijms26031042

Figure Lengend Snippet: Figure 6. Loss of PTPµ contributes to increase the affinity of α5β1 integrin toward OPN. (A) Total RNA was extracted from osteoblasts of bipedal wild-type (WT) and PTPµ −/−mice, and mRNA expression levels of CD44 and indicated beta (β) alpha (α) integrins were examined by qPCR analysis using β-actin as the internal control. Error bars show standard error of the mean of three independent experiments performed in duplicate. (B) WT and PTPµ −/−osteoblasts (samples from 3 mice per genotype were pooled (n = 3)) were lysed and 20 µg of proteins were resolved by 10% SDS-PAGE and immunoblotted for antibodies specific for the indicated proteins. OPN served as the loading control. (C) WT and PTPµ −/−osteoblasts were lysed and subjected to immunoprecipitation (samples from 3 mice per genotype were pooled (n = 3)) with antibodies directed against each integrin subunit as indicated, including CD44 and OPN, and immunoprecipitates were resolved by 10% SDS-PAGE. Western blots were revealed with an antibody raised against OPN. OPN served as the loading control. The molecular weights for the integrins β1, β3, β5, β8, α1, α4, α5, αv, CD44, OPN are respectively 138kDa, 125kDa, 100kDa, 97kDa, 150kDa, 150kDa, 150kDa, 135kDa, 81kDa and 66kDa. (D) MC3T3- E1 cells (mouse osteoblasts) treated with PBS or rOPN were subjected to immunoprecipitation with antibodies against the Gi1, Gi2, or Gi3 alpha subunit. Precipitates were resolved by 10% SDS-PAGE and immunoblotted with an antibody directed against phospho-serine. Cells were pre-treated with antibody against β1 integrin for 30 min followed by an 18 h incubation with 0.5 µg/mL rOPN, prior to immunoprecipitation and immunoblotting. Bands shown are representative of results obtained with independent experiments. A second replicative experiment is shown in Supplementary Figure S6.

Article Snippet: The immune complexes were analyzed by Western blot analysis with antibody against integrin β1 (SC-6622), integrin β3 (SC6627), integrin β5 (SC-5401), integrin β8 (SC-514150), integrin α1 (sc-271034), integrin α4 (sc-6589), integrin α5 (sc-166681), (Santa Cruz Biotechnology Inc.), or integrin αv (4711) (Cell Signaling Technology, Danvers, MA, USA).

Techniques: Expressing, Control, SDS Page, Immunoprecipitation, Western Blot, Incubation