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. 2018 Sep 10;9:521. doi: 10.3389/fendo.2018.00521

Figure 2.

Figure 2

C. elegans RNA Polymerase II is O-GlcNAcylated. (A) RNA Polymerase II (Pol II) was immunopurified from extracts prepared from starved (S) and fed (F) L1 larvae using wild type or O-GlcNAc cycling mutant strains. Western blots were probed with an anti-O-GlcNAc antibody (RL2) or anti-Pol II antibody that recognizes isoforms independent of CTD phosphoepitopes (8WG16), as indicated. The position of the major Pol II band is indicated by arrows in the blots. This band was quantified in each lane using ImageJ and the relative ratio of RL2 to 8WG16 signal intensity plotted in the graph above. The oga-1 mutant strain has dramatically increased levels of O-GlcNAcylated Pol II that is further increased upon feeding. (B) RNA Pol II was immunopurified from extracts prepared from starved (S) and fed (F) animals as in (A). A modified recombinant galactosyltransferase was used to introduce a GalNAz label to O-GlcNAc modified proteins (52). The GalNAz label was reacted with an alkyne-TAMRA probe using “Click” chemistry (53). The top row of Western blot images shows the anti-TAMRA antibody detection results. As a positive control, α-crystallin (20 pg) was detected using the same method. The second row of images shows the anti-Pol II signal (8WG16) from an identical blot; note the absence of signal for α-crystallin. The last row of images shows the overlay of signals from the two fluorophores corresponding to O-GlcNAc and Pol II. The position of the major Pol II band is indicated by arrows in all three blot images. This band was quantified in each lane using ImageJ and the relative ratio of TAMRA to 8WG16 signal intensity plotted in the graph above. (C) Pol II in oga-1 mutants has a higher molecular mass. The post-nuclear supernatants (see Experimental Procedures) from wild type and mutant strains were run on 10–20% SDS-PAGE gels and transferred for immunoblotting as described in Experimental Procedures. The primary antibody was the RNA Pol II Ser-2-P antibody ab5095 and the secondary IR-labeled anti-mouse IgG. The blot was scanned with an Oddysey IR scanner as described previously (25).