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. 2017 Dec 21;293(6):2219–2230. doi: 10.1074/jbc.RA117.000682

Figure 4.

Figure 4.

β1,4GalTV regulates the activity of Notch1 signaling. A, total lysates of T698968 cells expressing control or β1,4GalTV shRNA3 were analyzed with Notch1 antibody by Western blotting. The protein expression of GAPDH served as a loading control. B, the relative densities of NICD protein levels in A were quantified using densitometry. Values are normalized to that of T698968 cells expressing LacZ shRNA. Results are expressed as mean ± S.D. (n = 3; *, p < 0.05). C, Western blot analysis of nuclear NICD expression in T698968 cells expressing control or β1,4GalTV shRNA3. Sp1 served as a nuclear marker. D, RT-PCR analysis of Hes-1 expression in T698968 cells expressing LacZ shRNA + FLAG, β1,4GalTV shRNA3 + FLAG, β1,4GalTV shRNA3 + GalTV, or β1,4GalTV shRNA3 + GalTV (Y268G/W294G). E, the relative densities of Hes-1 PCR product levels were quantified using densitometry. Values are normalized to that of cells expressing LacZ shRNA + FLAG. Results are expressed as mean ± S.D. (n = 3; ***, p < 0.001). F, RT-PCR analysis of Notch1 expression in T698968 cells expressing control or β1,4GalTV shRNA. Actin expression served as a loading control. The relative densities of Notch1 PCR product levels were quantified using densitometry. Values are normalized to that of cells expressing control shRNA. **, p < 0.01. G, Western blot analysis of Notch1 from lysates of T698968 cells expressing control or β1,4GalTV shRNA in the absence or presence of the lysosome inhibitor chloroquine overnight. H, Notch1 proteins immunoprecipitated from T698968 cells expressing control or β1,4GalTV shRNA were resolved by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The membrane was overlaid with Notch1 antibody to verify the immunoprecipitation efficiency, with lectin RCA-1 to confirm β1,4-linked galactosylation, and with β1,4GalTV antibody to examine the interaction between β1,4GalTV and Notch1. IB, immunoblot. I, trafficking of newly synthesized Notch1 to the cell surface in T698968 cells expressing LacZ shRNA or β1,4GalTV shRNA3. Pulse labeling was performed with 1 mm AHA (a structural analog of methionine). The AHA-labeled Notch1 trafficked to the cell surface was examined using Western blotting. Results are expressed as percent of AHA-labeled Notch1 in cells expressing LacZ shRNA at 60 min. Data are represented as the means ± S.D. from three separate experiments. J, Notch1 proteins immunoprecipitated from T698968 cells expressing control or β1,4GalTV shRNA treated with His-tagged galectin-3 for 30 min were resolved by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The membrane was overlaid with Notch1 antibody to verify the IP efficiency and with His antibody to examine the interaction between His-tagged galectin-3 and Notch1. K, total lysates of T698968 cells expressing control or β1,4GalTV shRNA3 treated with or without His-tagged galectin-3 protein for 24 h were analyzed with Notch1 antibody by Western blotting. The protein expression of GAPDH served as a loading control. FL-Notch1, full-length Notch1.