JCS040691 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Quantitative western blotting of INF2 in Swiss 3T3 cells. 1 µg of Swiss 3T3 cell extract (corresponding to 1430 cells) was supplemented with the indicated ng amounts of recombinant mouse INF2-(C), resolved by SDS-PAGE, and blotted using antibody raised against mouse INF2-(C). Densitometry of the INF2 and INF2-(C) bands allowed calculation of 381,000±32,000 INF2 molecules/cell. Calculations of cell volume from diameters of trypsinized cells suggest that INF2 is present at approximately 300 nM in these cells.

• Supplemental Figure S2 -

  Fig. S2. Colocalization of INF2 with an endogenous ER protein. (A) Formaldehyde-fixed Swiss 3T3 cells were immunostained for INF2 (red) and the luminal ER chaperone, GRP94 (green). Since both antibodies were from rabbit, the anti-INF2 antibody was directly labeled with Cy3, and cells were treated with Cy3-anti-INF2 after having stained with anti-GRP94 and fluorescein-labeled anti-rabbit IgG. Images in lower row represent enlargements of the boxed region. (B) Cells were treated with 3 µM nocodazole for 30 minutes before fixation, then stained as in A. Independent experiments showed that this treatment resulted in complete microtubule depolymerization.

• Supplemental Figure S3 -

  Fig. S3. GFP-INF2 localizes to the reticular network. Fluorescence microscopy of a Swiss 3T3 cell expressing GFP-INF2 and mCherry-Sec61. Enlarged images shown below. Cells were electroporated with GFP-INF2 and mCherry-Sec61, plated on glass coverslips and fixed with formaldehyde 24 hours after transfection.

• Supplemental Figure S4 -

  Fig. S4. ER localization of Endogenous INF2 is stable to cell permeabilization. Swiss 3T3 cells were permeabilized with digitionin on ice for the times indicated on the figure, then fixed and stained for INF2 (antibody staining), polymerized actin (TRITC-phalloidin) and DNA (DAPI). All images for a given staining were taken for the same exposure time and processed identically.

• Supplemental Figure S5 -

  Fig. S5. Prenyl group identification by LC/MS-MS analysis of INF2 peptides. (A) I NF2 is both farnesylated and partially carboxymethylated at its C-terminus. Ion chromatograms from high mass accuracy Orbitrap MS1 spectra of tryptic peptides from immunoprecipitated INF2 digest. (i) Base peak and ion chromatograms for (ii) RLfC-OH+H⁺, 2H⁺⁺,²⁺, (iii) RLfC-OCH₃+H⁺, 2H⁺⁺,²⁺, (iv) LfC-OH+H⁺⁺, (v) LfC-OCH₃+H⁺⁺ extracted ±1.5 parts per million relative mass deviation from the theoretical exact masses of these species, where ‘f’ represents farnesyl group. Insets show elution-time-averaged MS1 spectra (with magnified regions described above brackets) corresponding to the retention times in the extracted chromatograms. Numbers in parentheses indicate each chromatogram base peak intensity in arbitrary units. (B) Tandem mass spectrometry confirms the presence of RLfC-OH and RLfC-OCH₃. Reciprocal ion trap tandem mass spectra corresponding to the (i) free acid (RLfC-OH) and (ii) methyl ester (RLfC-OCH₃) of farnesylated C-terminal tryptic peptides from INF2. Characteristic fragment ions are highlighted on the respective structures. (C) Tandem mass spectrometry confirms the presence of LfC-OH and LfC-OCH₃. Reciprocal ion trap tandem mass spectra corresponding to the (i) free acid (LfC-OH) and (ii) methyl ester (LfC-OCH₃) of farnesylated C-terminal tryptic peptides from INF2. Characteristic fragment ions are highlighted on the respective structures.

• Supplemental Figure S6 -

  Fig. S6. Expression of GFP-INF2-WH2/DAD mutant causes collapse of ER around nucleus. Swiss 3T3 cells were transfected with either GFP-INF2-W, alone (A,C) or with mCherry-Sec61 (B), fixed in formaldehyde 24 hours after transfection, stained with DAPI (A,B) or DAPI+TRITC-phalloidin (C), and imaged. No peripheral reticular staining for GFP-INF2 is detected, and mCherry-Sec61 colocalizes with the tubular structures, suggesting ER collapse. In C, images are over-processed to show the absence of actin filaments or GFP-INF2-W in peripheral regions. 100% of cells with collapsed ER tubules also had actin accumulation (n=200 cells from three experiments).

• Movie 1 -

  Movie 1. Live-cell imaging of GFP-INF2 and mCherry-Sec61. Swiss 3T3 cells cotransfected with the two expression plasmids were examined live and images acquired in the green (INF2) and red (Sec61) channels alternately at 15 second intervals for 5 minutes. Loss of signal over time is due to rapid photo-bleaching.