Fig 1: EDEM3 function in the presence of Man1B1. (A) Western blot comparison of NHK processing by EDEM3 constructs in EDEM3-KO and MAN1B1-OE cell lines. After co-transfection of indicated plasmids, cell lysates were separated by SDS-PAGE and processed for WB with rabbit anti-A1AT, rat anti-HA, mouse anti-MAN1B1 and mouse anti-actin antibodies. (B) Western blot comparison of ST processing by EDEM3 constructs in EDEM3-KO and MAN1B1-OE cell lines. Cells were co-transfected with corresponding plasmids and analyzed by WB with mouse anti-TYR, rat anti-HA, mouse anti-MAN1B1. Rat anti-tubulin (TUB) were used as loading control. (C) Immunolabeling analysis of intracellular NHK in EDEM3-KO and MAN1B1-OE cell lines. Cells were co-transfected with NHK and WT or MAN plasmids and they were pulse-labeled accordingly. After immunoprecipitation, the isolated complexes were separated by SDS-PAGE and results were visualized by autoradiography. Graphic representation of NHK degradation rate is presented as (mean ± SEM) of 3 independent experiments. (D) Extracellular fate of NHK in EDEM3-KO and MAN1B1-OE cells. Collected cell media from (C) was processed for immunoprecipitation with anti-A1AT antibodies and the obtained immunocomplexes were separated by SDS-PAGE. NHK secretion rate is represented as (mean ± SEM) of three independent experiments. (E) ST processing by WT and MAN in EDEM3-KO and MAN1B1-OE cell lines. Cells were immunolabeled as described and subjected to immunoprecipitation with rabbit anti-tyrosinase (TYR) antibodies. Immunocomplexes were separated by SDS-PAGE and the results were visualized by autoradiography.
Fig 2: (A) EDEM3 constructs are processed by EndoH. EDEM3-KO cells were transfected with plasmids encoding for EDEM3 constructs. Lysates were treated with EndoH and subjected to Western blotting. (B) Same as in (A), except lysates were also incubated with PNG-aseF. (C) Western bloting analysis of NHK processing by EDEM3 constructs in HEK293T cell line. After co-transfection with NHK and EDEM3 constructs, cells were lysed and processed for WB. Rabbit anti-A1AT and rat anti-HA antibodies were used for detection. Actin was used as loading control. (D) NHK intracellular processing in EDEM3-KO and MAN1B1-OE cells. Following co-transfection with the appropriate plasmids, cells were starved, pulse labeled and chased for the indicated time points. Immunoprecipitation was performed using rabbit anti-A1AT antibodies. Resulting immunocomplexes were separated by SDS-PAGE and results were visualized by autoradiography. (E) Same as in (D) with the exception that ST was used.
Supplier Page from DNASU for MAN1B1 (Homo sapiens) in pDONR221 (Gateway donor/master vector)