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. 2015 Feb 4;16(3):332–340. doi: 10.15252/embr.201439123

Figure 5.

Figure 5

Reptin silencing enhances ATF6 and XBP1s activation
  • A GRP78 and GRP94 expression was detected using anti-KDEL antibodies (top blot) in HuH7 cells treated or not with tunicamycin and/or doxycycline (Dox) to induce reptin silencing (bottom blot). Expression of p97 was also monitored (middle blot).
  • B eIF2α phosphorylation was monitored using specific antibodies (top blot) and reported to the total expression (bottom blot) in HuH7 cells treated or not with tunicamycin and/or doxycycline (Dox).
  • C ATF6 activation was monitored in the same experimental conditions using antibodies against the N-terminal domain of ATF6.
  • D Expression of unspliced XBP1 mRNA as determined by RT–PCR and expression of the XBP1s ligase RTCB as determined by immunoblot using anti-RTCB antibodies in HuH7 cells treated or not with tunicamycin and/or doxycycline (Dox).
  • E XBP-1 mRNA splicing as determined by RT–PCR under basal conditions or upon tunicamycin treatment (5 μg/ml for 16 h) in HuH7 cells subjected or not to doxycycline-induced Reptin silencing. Three independent experiments were performed, and a representative image is shown.
  • F HuH7 cells were treated either with DBeQ (20 μM, D), tunicamycin (2 μg/ml; T) or both for 4 h. XBP-1 mRNA splicing was determined by RT–PCR (Mean ± SD,N + 3). P-values were calculated using multiple t-test corrected using the Holm-Sidak method. *P < 0.05; **P < 0.01.
  • G HuH7 cells were treated with tunicamycin (2 μg/ml; T) for 1 h. XBP1s was immunoprecipitated, and the complex was immunoblotted with anti-Reptin antibodies (top blot). Total cell lysate (TCL) was immunoblotted with anti-Reptin (middle blot) or anti-XBP1s (bottom blot).