Figure 2. Hydroxylase P4HA2 forms a complex with Carabin and downregulates Carabin. (A) Carabin exotically interacts with P4HA2. 293T cells were transfected with Flag-Carabin. Cell lysates were prepared and subjected to immunoprecipitation with (M2) anti-Flag beads. The immunoprecipitates were analyzed by WB with anti-Flag, P4HA2, P4HB, and calcineurin antibodies. (B) Carabin combines with P4HA2 in vivo. SU-DHL-6 cell lysates were incubated with protein A/G-Sepharose with either anti-Carabin or control IgG antibody. The immunoprecipitates were resolved by SDS-PAGE and followed by WB with anti-Carabin and anti-P4HA2 antibodies. (C) Schematic diagram of truncated GST-Carabin. Various truncated mutation constructs of GST-Carabin are shown schematically. (D) Mapping the interaction domain of Carabin with P4HA2. Purified GST fusion protein of Carabin WT (a) or mutants (b-e) were bound to glutathione-Sepharose beads and incubated with lysates of 293T cells transfected with the V5-P4HA2 expression construct. Cell lysates (CL) and GST pulldown immunoprecipitates were analyzed by WB. (E) Negative relevant expression of Carabin and P4HA2 in B-lymphoma cell lines. Detection protein expression of Carabin and P4HA2 in DB, BC2, SU-DHL-4, SU-DHL-6, OCI-Ly3 NU-DUL-1, and SU-DHL-2 B-lymphoma cell lysates. 293T are the positive controls of P4HA2. (F) Positive correlation between P4HA2 expression and DLBCL cell proliferation. Cell growth assays were analyzed as described above. Points, mean (n = 6); bar, SD. (G) Overexpression of P4HA2 leads to degradation of Carabin in a dose-dependent manner. The same amount of Flag-Carabin was cotransfected with increasing amounts of V5-P4HA2 plasmids into 293T cells. The protein levels of Flag-Carabin and V5-P4HA2 were detected by WB. (H) Carabin protein is upregulated by knockdown of P4HA2 in B-lymphoma cells. SU-DHL-4, SU-DHL-6, and NU-DUL-1 cells of P4HA2 knockdown were analyzed Carabin expression by WB. (I) Quantification of Carabin upon P4HA2 knockdown. Columns, mean (n = 3); bar, SD; *P < .05; **P < .01; ***P < .001. NC, control cell; shP1, cells stably expressing P4HA2-specific shRNA1; shP2, cells stably expressing P4HA2-specific shRNA2.
Figure 2.

Hydroxylase P4HA2 forms a complex with Carabin and downregulates Carabin. (A) Carabin exotically interacts with P4HA2. 293T cells were transfected with Flag-Carabin. Cell lysates were prepared and subjected to immunoprecipitation with (M2) anti-Flag beads. The immunoprecipitates were analyzed by WB with anti-Flag, P4HA2, P4HB, and calcineurin antibodies. (B) Carabin combines with P4HA2 in vivo. SU-DHL-6 cell lysates were incubated with protein A/G-Sepharose with either anti-Carabin or control IgG antibody. The immunoprecipitates were resolved by SDS-PAGE and followed by WB with anti-Carabin and anti-P4HA2 antibodies. (C) Schematic diagram of truncated GST-Carabin. Various truncated mutation constructs of GST-Carabin are shown schematically. (D) Mapping the interaction domain of Carabin with P4HA2. Purified GST fusion protein of Carabin WT (a) or mutants (b-e) were bound to glutathione-Sepharose beads and incubated with lysates of 293T cells transfected with the V5-P4HA2 expression construct. Cell lysates (CL) and GST pulldown immunoprecipitates were analyzed by WB. (E) Negative relevant expression of Carabin and P4HA2 in B-lymphoma cell lines. Detection protein expression of Carabin and P4HA2 in DB, BC2, SU-DHL-4, SU-DHL-6, OCI-Ly3 NU-DUL-1, and SU-DHL-2 B-lymphoma cell lysates. 293T are the positive controls of P4HA2. (F) Positive correlation between P4HA2 expression and DLBCL cell proliferation. Cell growth assays were analyzed as described above. Points, mean (n = 6); bar, SD. (G) Overexpression of P4HA2 leads to degradation of Carabin in a dose-dependent manner. The same amount of Flag-Carabin was cotransfected with increasing amounts of V5-P4HA2 plasmids into 293T cells. The protein levels of Flag-Carabin and V5-P4HA2 were detected by WB. (H) Carabin protein is upregulated by knockdown of P4HA2 in B-lymphoma cells. SU-DHL-4, SU-DHL-6, and NU-DUL-1 cells of P4HA2 knockdown were analyzed Carabin expression by WB. (I) Quantification of Carabin upon P4HA2 knockdown. Columns, mean (n = 3); bar, SD; *P < .05; **P < .01; ***P < .001. NC, control cell; shP1, cells stably expressing P4HA2-specific shRNA1; shP2, cells stably expressing P4HA2-specific shRNA2.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal