JCS033399 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. No detectable endogenous is EGFR expressed in CHO cells. CHO cells were transfected with (left) or without (right) expression plasmids for EGFR-VN and EGFR-VC were treated with 100 ng/ml EGF for 30 minutes at 4°C. Whole cell lysates were subjected to immunostaining with anti-EGFR antibody, anti-phospho-EGFR antibody and anti-α-tubulin antibody.

• Supplemental Figure S2 -

  Fig. S2. Subcellular localization of the ErbB receptors. CHO cells were co-transfected with a pair of expression plasmids coding for FP-fused receptors as indicated. The cells were serum-starved, and then observed by confocal microscopy. Bars, 10 µm.

• Supplemental Figure S3 -

  Fig. S3. Examination of receptor protein expression levels in the BiFC-positive and -negative controls by western blotting. CHO cells expressing the BiFC constructs indicated were serum-starved, and whole cell lysates were subjected to immunoblotting using anti-FLAG antibody for the detection of FLAG-tagged receptors and anti-HA antibody for the detection of HA-tagged receptors.

• Supplemental Figure S4 -

  Fig. S4. Ligand binding does not affect the fluorescence intensities of cells expressing BiFC construct heterodimers. CHO cells co-expressing the BiFC constructs indicated were serum-starved and then incubated with ATP synthesis inhibitors for 1.0 hour to block endocytosis, as described in Materials and Methods. Fluorescence intensities of ROI on the cell membrane were observed by confocal microscopy. 100 ng/ml EGF or 1.0 nM NRG was added to the cell culture at the time indicated by arrow. The relative intensities were normalized by the average intensity at 3 minutes,. Data points are the means ± s.d.

• Supplemental Figure S5 -

  Fig. S5. The ErbB receptor homo- and heterodimers are formed in ER. (A) NIH3T3 cells were co-transfected with a pair of expression plasmids coding for the BiFC constructs indicated. The cells were serum-starved and treated with (+) 10 µg/ml BFA for 8 hours at 37°C. The cells were fixed with methanol-acetone and immunostained with anti-calnexin antibody, followed by incubation with Cy3-conjugated secondary antibody. Nuclei were also stained with Hoechst 33342. Fluorescence signals for calnexin (red), Venus (green) and Hoechst 33342 (cyan) were observed by confocal microscopy. (B) Confirmation of the expression of BiFC fusion constructs in the negative control cells. NIH3T3 cells co-transfected with BiFC constructs encoding the receptor-VN indicated and EpoR-HA-VC were treated with 10 µg/ml BFA for 8 hours at 37°C, fixed with methanol-acetone, and immunostained with anti-ErbB primary antibodies indicated and anti-HA primary antibody for EpoR-HA-VC, followed by incubation with Cy3-conjugated donkey secondary antibody and Alexa Fluor 633-conjugated antibody. Nuclei were also stained with Hoechst 33342. The cells were observed by confocal microscopy. Note that the ErbB receptors and EpoR were clearly expressed in ER, but no Venus fluorescence was observed. Bars, 10 µm.

• Supplemental Figure S6 -

  Fig. S6. Nuclear localization of ErbB3-GFP. (A) CHO cells were transiently transfected with an expression plasmid for unfused GFP or ErbB3-GFP. After being serum-starved for 24 hours, the cells were observed by confocal microscopy. Note that GFP (left) was uniformly distributed throughout the nucleus and cytoplasm, but ErbB3-GFP fusion proteins (middle and right) were mainly localized in the nucleus and minor fractions in the cytoplasm irrespective of its expression levels. Bars, 10 µm. (B) CHO cells were transfected with an expression plasmid for GFP or ErbB3-GFP and then serum-starved for 24 hours. Whole cell lysates were subjected to immunostaining using an anti-GFP antibody (1:200 dilution; Santa Cruz Biotechnology; sc-8334) for the detection of GFP or ErbB3-GFP. Note that ErbB3-GFP proteins were not cleaved.