Figure 7

The level of Rab8 (A) and TfR (B) co-precipitated with optineurin-GFP (OPTN-GFP). RGC5 cells were transfected with pEGFP-N1, and GFP-tagged wild type, E50K, R96L, Q398X, and E478G optineurin constructs. OPTN-GFP was immunoprecipitated with anti-GFP microbeads. Pulled down protein was immunoblotted with anti-GFP to verify the immunoprecipitation (IP) procedure. Co-precipitated proteins were immunoblotted (IB) with anti-Rab8 (A), anti-TfR (B), or anti-GFP antibody. Densitometry was performed to quantify the intensity of Rab8, TfR and OPTN-GFP bands. The values were normalized against OPTN-GFP and the ratios between the mutants and wild type optineurin are presented. Note that the levels of Rab8 and TfR co-precipitated with OPTN-GFP in the mutants were similar to, or higher than, those in the wild type. As was expected, no Rab8 or TfR protein co-precipitation with GFP (25 kDa, asterisks) was detected in lysates collected from cells transfected with EGFP-N1 empty vector (C). The levels of Rab8 and TfR co-precipitated with optineurin. RGC5 cells were non-transfected, or transfected with pEGFP-N1, or GFP-tagged wild type or E50K optineurin construct. Optineurin-GFP (OPTN-GFP) and endogenous optineurin (OPTN) were immunoprecipitated with anti-optineurin polyclonal antibody. Non-transfected RGC5 cell lysate was immunoprecipitated with normal IgG as negative control. The pulled down protein was immunoblotted with anti-Rab8 (top panel), anti-TfR antibody (middle panel) or anti-optineurin antibody (bottom panel, for verification of the IP procedure). Normal IgG did not yield any band for TfR or Rab8 as was expected. The ratios of Rab8/endogenous OPTN (top panel) and TfR/endogenous OPTN (middle panel) from wild type and E50K optineurin-GFP-expressing cells were higher than those of GFP control.