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. 2011 Aug 8;157(2):730–741. doi: 10.1104/pp.111.179861

Figure 2.

Figure 2.

Rapamycin induces Thr phosphorylation of the BiP chaperone in its peptide-binding domain. A, Phosphorylation time course of the 70-kD protein following rapamycin treatment. Rapamycin (500 nm) or drug vehicle was added to wild-type (wt) or rapamycin-insensitive (rap2) Chlamydomonas cells, and samples were collected at the indicated times. Soluble extracts were fractionated by SDS-PAGE and analyzed by western-blotting, probing with phospho-Thr (P-Thr), BiP, and HSP70B antibodies. B, 2D western-blot analysis of BiP and HSP70B. Wild-type Chlamydomonas cells were treated with 500 nm rapamycin for 12 h (rap), heat shocked at 40°C for 1 h (HS), or remained untreated (control). Soluble extracts were resolved by 2D electrophoresis as described in “Materials and Methods” using pH 4 to 7 strips, and gels were processed for immunodetection with phospho-Thr (P-Thr), BiP, and HSP70B antibodies. IEF, Isoelectric focusing.