Xenbase Image ID: 125147

Figure 1. CLNX is dephosphorylated during ER stress by CN.(A) IPs of [γ32P]ATP-labeled CLNX from oocytes in the absence (0 minutes) or presence (15, 30 and 60 minutes) of Tg (1 µM) were performed. The samples were resolved through 12% SDS-PAGE, transferred to nitrocellulose and P-CLNX visualized by autoradiography (top panel). For loading control, a Western blot of CLNX was performed in oocyte microsomal extracts before the IPs (bottom panel). Histogram depicts the relative intensity of each band relative to the corresponding density of the CLNX Western blot. Notice that exogenous CLNX is expressed at higher levels than endogenous CLNX and that its autoradiographic signal is significantly higher than the signal from endogenous levels of phosphorylatioed CLNX (Figure S1). (B) Immunodetection by Western blotting of control oocytes and CLNX overexpressing oocytes. Top panel shows endogenous and exogenous CLNX. Middle panels show phosphorylated eIF2α (P-elF2α) and BiP (Assay Designs cat# SPA-826) in each corresponding cytosolic fraction. Lower panel shows α-actin loading controls. (C) Samples from Tg-treated oocytes that were pre-incubated CsA (200 nM) and FK506 (20 nM) for 16 hours are presented in lane 3. Immunodetection of CLNX by Western blotting was used as a loading control (lower panels). Histogram depicts the mean intensity of each band relative to the corresponding density in the Western blots of overexpressed CLNX. DMSO (0.05% v/v) is used as the vehicle control. Notice that control oocytes injected only with CsA/FK506 do not exhibit increased stress as indicated by Western blot analysis of eIF2α -P or BiP (Figure S2). (D) Samples from Tm-treated oocytes (lanes 2 and 4) that were pre-incubated or not with inhibitors CsA and FK506 as indicated above are shown in lanes 3 and 4. The middle panels show Western blots of CLNX of the oocyte microsomal extracts before IPs. Histogram shows the relative intensities of P-CLNX compared to overexpressed CLNX. Methanol (0.05%v/v) is used as the vehicle control. Data represents 3 independent experiments with 10 oocytes per group. Image published in: Bollo M et al. (2010) Bollo et al. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution license Larger Image Printer Friendly View

Return to previous page