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. Author manuscript; available in PMC: 2010 Mar 30.
Published in final edited form as: Immunity. 2007 Feb 1;26(2):215–226. doi: 10.1016/j.immuni.2006.12.005

Figure 4. gp96 Is a Molecular Chaperone for TLR9.

Figure 4

(A) The WT and gp96-deficient F4/80+ cells were analyzed for cell-surface expression of TLR2 and TLR4 (open histogram). Shaded histogram represents isotype control.

(B) Comparative TLR mRNA level in WT and KO BMDM by Q-PCR. Number below represents number of threshold cycle (average of 2 duplicate runs).

(C) IB analysis of TLR9, gp96, and β-actin in WT and KO cells.

(D) Confocal analysis of TLR9 and gp96 expression in WT and KO BMDMs. Day +3 WT or KO BMDMs were transduced with a retroviral vector expressing TLR9-HA and enhanced GFP (EGFP) in the bicistronic fashion. 2 days later, cells were seeded and cultured on glass coverslips, fixed, permeablized, blocked, and double stained with mouse anti-HA Ab and rat anti-gp96 Ab, followed by goat anti-mouse Alexa Fluor 647 (TLR9, red) and goat anti-rat Alexa Fluor 488/Rhodomine Red (gp96, green) secondary Ab. Images were captured with a Zeiss LSM 510 Axiovert 100 confocal microscope equipped with an argon/krypton laser.

(E) Confocal analysis of TLR9 expression. HEK293-TLR9 cells were cultured on glass coverslips, fixed, permeablized, and stained for gp96 (green) and TLR9 (red). The nucleus was counterstained with PI (blue). The merged images (gp96 and TLR9, light transmission and PI) were also shown.

(F) gp96 is coprecipitated with TLR9. HEK293-TLR9 cells were immunoprecipitated with HA Ab or isotype control Ab, treated with nothing, PNGase F, or Endo H, followed by IB for gp96 and TLR9.

(G) Total cell lysates of HEK293-TLR9 were immunoblotted for gp96, developed with substrates of either high sensitivity (HS) or low sensitivity (LS).

(H) HEK293-TLR9 cells or WT BMDM were immunoprecipitated with 9G10, SPA851, or respective isotype control Ab, followed by SDA-PAGE and IB for gp96 and TLR9.

(I) HEK293-TLR9 cells were treated with buffer only, tunicamycin, thapsigargin, or antimycin A. Cells were then immunoprecipitated for TLR9 followed by IB for gp96; the aliquot of the total cell lysates were immunoblotted for gp96, TLR9, and β-actin.