Fig. 3.

N-glycosylation regulates TMIGD1 localization at cell-cell contacts. a Lysates obtained from Flag-TMIGD1-expressing HEK293T cells were incubated with PNGaseF, separated by SDS-PAGE and analyzed by Western blotting with antibodies against the Flag tag. Note that the electrophoretic mobility of TMIGD1 shifts to a M_r that corresponds to the M_r expected for the unglycosylated protein, indicating that TMIGD1 is decorated exclusively with N-linked carbohydrates. b Lysates obtained from MDCKII cell lines expressing Flag-tagged TMIGD1 constructs (TMIGD1/WT (−/WT), TMIGD1/Δ5 (−/Δ5), ΔD1-TMIGD1 (ΔD1-), ΔD1-TMIGD1/Δ5 (ΔD1−/Δ5) were incubated with PNGaseF and analyzed by Western blotting with antibodies against the Flag tag. The electrophoretic mobilities of all TMIGD1 constructs shift to M_r that correspond to the M_r expected for the unglycosylated proteins. Note that the absence of the D1 domain (ΔD1, ΔD1−/Δ5 constructs) results in a protein species with a M_r similar to that of the full length proteins (/WT, /Δ5, respectively). These protein species most likely result from hyperglycosylation in the absence of the D1 Ig-like domain. c MDCKII cell lines described in (b) were incubated for 20 h with various concentrations of tunicamycin. Lysates were analyzed by Western blotting with antibodies against the Flag tag. Arrowheads indicate the TMIGD1 isoforms in the absence of tunicamycin treatment, asterisks indicate the TMIGD1 isoforms expected from the unmodified proteins. Protein bands above and below the protein bands marked by asterisks most likely represent incompletely deglycosylated protein species and degradation products due to the lack of glycosylation, respectively. d ΔD1-TMIGD1-expressing MDCKII cells were fixed with methanol and double-stained with antibodies against the Flag tag and against β-catenin. Flag-ΔD1-TMIGD1 is completely absent from cell-cell contacts after treatment with 0.5 μg/ml tunicamycin. Abbreviations: Dox., doxycycline, TM, tunicamycin, α-tub, α-tubulin. Scale bars: 10 μm