Endocytic Trafficking Routes of Wild Type and F508 Cystic Fibrosis Transmembrane Conductance Regulator

Supplemental Material

This article contains the following supporting material:

• Supplemental Table.pdf -
  Quantification of Co-localization of CFTR with different intracellular markers as shown in Figure 3. The co-localization of CFTR with sub-cellular marker proteins as shown in Figure 3 was quantified as described by Setiadi et al. (1998). Briefly the green and red images were converted to binary images. Pixels were then counted using the ImageJ 1.30v software (Wayne Rasband, National Institutes of Health, Bethesda, MD, http://rsb.info.nih.gov/ij/) in individual panels and a panel created by multiplying them.
  
• Supplemental Figure 1.pdf -
  Staining patterns of Extope-CFTR are similar with 16B12 IgGs or Fab fragments. (A) Staining pattern of Extope-CFTR using 16B12 IgGs or Fab fragments were similar when detected by goat anti-mouse Fab specific IgG (Jackson Immuno Research Labs.), followed by donkey anti-goat IgG Alexa Fluor 488 conjugate (Molecular Probes). Cells were pre-cooled and labeled for 30 minutes on ice and then washed and re-incubated for indicated times.

  (B) Staining pattern of Extope-CFTR with Fab fragments and IgGs detected with polyclonal goat anti-mouse IgG Alexa Fluor 488 conjugate. Cells were pre-cooled and labeled for 30 minutes on ice with 16B12 IgG or Fab and then washed and re-incubated for indicated times. Staining with Fab is much weaker when detected with polyclonal goat anti-mouse IgG Alexa Fluor 488 conjugate as compared to IgG.
  

• Supplemental Figure 2.pdf -
  PBS pH 3.7 removes external labeling of Extope-CFTR with 12CA5 mAb. Cell surface Extope-CFTR was labeled on pre-cooled, intact cells for 30 minutes on ice with monoclonal mouse antibodies 16B12 or 12CA5. Subsequently cells were washed with ice-cold PBS pH 7.4 followed by 2 washes with either PBS pH 3.7 (Acidic wash) or pH 7.4 (no acidic wash). Subsequently cells were washed 1X with PBS pH 7.4, fixed for 10 minutes with 4% paraformaldehyde and immunostained with goat anti-mouse IgG Alexa Fluor 488 conjugate as described in Methods.
  
• Supplemental Figure 3.pdf -
  Quantification of experiments shown in Figure 2A: Cell surface pools of Extope-CFTR are internalized. Ratio of fluorescence associated with plasma membrane to intracellular pools was determined using ImageJ 1.30v software (Wayne Rasband, National Institutes of Health, Bethesda, MD, http://rsb.info.nih.gov/ij/). Each point represents the average of at least 10 cells and standard deviations are indicated.

  Quantification of experiments shown in Figure 2B: Internalized pools of CFTR are rapidly recycled. Internal pools of Extope-CFTR were labeled with 12CA5 mAb and the ratio of fluorescence associated with plasma membrane to intracellular pools at different times determined using ImageJ 1.30v software. Each point represents the average of at least 10 cells and standard deviations are indicated.
  

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