(A) Sequences of ICL3 in other species from fish to primates. Asterisks indicate the position of two class II mutations: S945L and D979A. Identity of amino acids between the different proteins are boxed in black, conserved residues are boxed in dark gray and semi-conserved substitutions in light gray. (B) Representative gels for the same co-immunoprecipitation experiments in HT-29 cells expressing endogenous CFTR and COMMD1. Lysates from HT-29 cells were immunoprecipitated (IP) with either 0.8 µg of anti-COMMD1 mAb (Abnova), 0.8 µg of anti-CFTR mAb (MAB25031, R&D Systems) or with 0.8 µg anti-mouse IgG as a control. Each immunoprecipitation sample was then split in half and loaded onto an 8% SDS-PAGE for CFTR detection and 11% SDS-PAGE for COMMD1 detection. Both gels were transferred to PVDF membrane and subjected to immunoblotting (IB). The 8% SDS-PAGE membrane was probed with anti-CFTR mAb (MM13-4) and the 11% SDS-PAGE membrane with a rabbit anti-COMMD1 pAb (Proteintech Group). Both membranes were probed with anti-α-tubulin as control (11% gel is shown). Filled and empty arrowheads indicate the fully- (170 kDa) and core-glycosylated (140 kDa) CFTR, respectively. * indicates mouse IgG light chain from the antibody used for immunoprecipitation. (C) COMMD1 constructions in pcDNA3.1/Topo plasmid. Two COMMD1 constructs were generated by adding a Myc-tag at the N-terminus of COMMD1: Myc-COMMD1 and a construct with a deletion of the COMM domain named Myc-COMMD1ΔCOMM. (D) Representative gels for the same co-immunoprecipitation experiment between COMMD1 and wt- in heterologous system. HeLa cells stably expressing wt- (spTCF-wt) or empty CFTR vector (spTracer) as control were transfected with Myc-COMMD1. spTCF-wt were transfected with Myc-COMMD1ΔCOMM. Lysates from all these experiments were subjected to SDS-PAGE, as in (B) after CFTR IP. The 8% SDS-PAGE membrane was probed with anti-CFTR mAb and the 11% SDS-PAGE membrane with anti-c-Myc mAb. Both membranes were probed with anti-α-tubulin as control (11% gel is shown).

(A) Cartoon indicating the location of the different markers (red), CFTR (violet) processing throughout the cell and the intracellular localization of COMMD1 (green). (B) Double-labeling studies with organelle markers were performed in stably expressing wt-CFTR HeLa cells. Immunostaining was performed on fixed and permeabilized cells using rabbit primary antibody (anti-COMMD1 pAb 1∶100) followed by Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody (green), and each organelle marker was labeled with mouse primary antibodies (calnexin 1∶400, 58k Golgi 1∶100, Rab4 1∶100, Rab11 1∶100, EHD1 1∶100, TfR 1∶200) followed by Alexa Fluor 555-conjugated goat anti-mouse secondary antibody (red). Non-immune anti-mouse and anti-rabbit controls were conducted to remove non-specific signals. Fluorescence images of cells were captured and analyzed with a Zeiss Axio Observer Z.1 confocal microscope with x63 objective. Scale bars: 10 µm.

(A) HeLa cells were transfected with CFTR constructs (wt, K946R, K951R or K978R-CFTR) and Myc-COMMD1 or empty vector as control (mock). The same quantities of lysates were immunoprecipitated with anti-CFTR mAb (MAB25031). Representative gels for the same experiment where each immunoprecipitation sample was then split in half and loaded onto two 8% SDS-PAGEs for CFTR detection and ubiquitin detection. Both gels were transferred to PVDF membrane and subjected to immunoblotting (IB). Lysates were loaded onto an 11% SDS-PAGE and sequential probing of the membrane was performed (COMMD1, α-tubulin and lastly ubiquitin). Filled and empty arrowheads indicate the fully- (170 kDa) and core-glycosylated (140 kDa) CFTR, respectively. (B) Quantification of ubiquitinated CFTR. Ratio of ubiquitinated CFTR to total CFTR in cells transfected with Myc-COMMD1 compared to the same ratio in mock-transfected cells was reported as 100% for each independent experiment. The means ± S.D. were obtained from five to three independent experiments.* P<0.05 is determined by t-test. (C) Representative gels for the same co-immunoprecipitation experiment between COMMD1 and wt, K946R, K951R or K978R-CFTR in heterologous system. HeLa cells were co-transfected with Myc-COMMD1 or empty vector (mock) and wt, K946R, K951R or K978R-CFTR. Lysates from all these experiments were subjected to SDS-PAGE after CFTR IP. The 8% SDS-PAGE membrane was probed with anti-CFTR mAb and the 11% SDS-PAGE membrane with anti-c-Myc mAb. Both membranes were probed with anti-α-tubulin as control (11% gel is shown). (D) Proposed model of COMMD1 interaction through its COMM domain with the N-terminal end of ICL3 to modulate CFTR ubiquitination.

(A) HeLa cells stably expressing wt-CFTR were transiently transfected with an empty COMMD1 vector (mock, pcDNA3.1/Topo) or Myc-COMMD1, and were biotinylated with Sulfo-NHS-LC-biotin. Lysates from all these experiments were subjected to SDS-PAGE directly (input) or pulled-down with streptavidin-agarose (biotin). Representative gels for the same samples were separated by 8% SDS-PAGE for CFTR, Na/K-ATPase detection and 11% SDS-PAGE for COMMD1, α-tubulin detection. (B) HeLa cells stably expressing wt-CFTR were transiently transfected with a siCONTROL Non-Targeting siRNA (mock) or COMMD1 siRNA and further processed as in (A). Filled and empty arrowheads indicate the fully- (170 kDa) and core-glycosylated (140 kDa) CFTR, respectively. (C) Quantification of CFTR cell surface expression. The biotinylated CFTR level is normalized to the biotinylated Na/K-ATPase level. Endogenous COMMD1 expression is referred as 100%, with mock being pcDNA3.1/Topo for overexpression experiments (A), whereas mock was siCONTROL for silencing experiments (B). The means ± S.D. were obtained from three independent experiments.* P<0.05 was determined by t-test. (D) Immunofluorescence microscopy of COMMD1 and CFTR in HeLa cells stably expressing wt-CFTR. Cells were transfected with Myc-COMMD1 or COMMD1 siRNA for overexpression and silencing studies, respectively, and not transfected for endogenous expression studies. Two types of light exposure microscopy (short and normal) are shown to visualize all expression conditions. Scale bars: 10 µm.

(A) Representative gels for the same CFTR IP experiment with MAB25031 from HeLa cells stably expressing wt-CFTR and separated on 8% SDS-PAGE transferred to PVDF membrane. Half of the membrane was probed with anti-CFTR mAb and the other half with anti-ubiquitin mAb. Lysates were loaded onto an 11% SDS-PAGE and sequential probing of the membrane was performed (COMMD1, α-tubulin and lastly ubiquitin). Filled and empty arrowheads indicate the fully- (170 kDa) and core-glycosylated (140 kDa) CFTR, respectively. (B) Quantification of ubiquitinated CFTR. Ratio of ubiquitinated CFTR to total CFTR in each condition is shown, endogenous COMMD1 expression is referred as 100%. The means ± S.D. were obtained from five independent experiments.* P<0.05 was determined by t-test. (C) Stability of the mature wt-CFTR was determined upon inhibition of protein biosynthesis with cycloheximide (CHX). Cells were incubated in the presence of cycloheximide for the indicated time intervals. (D) Quantification of mature CFTR was normalized to α-tubulin level.