PGAM1/2/4 Antibody

Western Blot: PGAM1/2/4 Antibody [NB100-774] -

Validation of R-Ras2 as the defatty-acylation target of SIRT6.(A) mRNA and protein levels of R-Ras2 in Sirt6 WT and KO MEFs. (B) In-gel fluorescence (with NH2OH treatment) showing that R-Ras2 has higher lysine fatty acylation level in Sirt6 KO MEFs than in Sirt6 WT MEFs. Right histogram shows the quantification of bands on the fluorescence gel. Values with error bars indicate mean ± s.d. of three biological replicates. * indicates p<0.05. The full fluorescence gel is shown in Figure 2—figure supplement 2A. (C) Detection of R-Ras2 lysine fatty acylation levels in control and SIRT6 knockown HEK 293 T cells by in-gel fluorescence. Right histogram shows the quantification of bands on the fluorescence gel. Values with error bars indicate mean ± s.d. of three biological replicates. * indicates p<0.05. (D) Lysine fatty acylation levels of endogenous R-Ras2 in Sirt6 WT and KO MEFs. (E, F) SIRT6 defatty-acylated R-Ras2 in a NAD+-dependent manner in vitro. In-gel fluorescence was used to detect R-Ras2 lysine fatty acylation (E). A 32P-NAD+ assay was used to detect fatty acyl ADPR product from defatty-acylation reaction. (F). (G) In-gel fluorescence (with NH2OH treatment) showing that mutation of four lysine residues at the C-terminus of R-Ras2 significantly decreased lysine fatty acylation in Sirt6 KO MEFs. Right histogram shows the quantification of bands on the fluorescence gel. Values with error bars indicate mean ± s.d. of three biological replicates. * indicates p<0.05. The full fluorescence gel including R-Ras2 total fatty acylation levels (without NH2OH treatment) is shown in Figure 2—figure supplement 2C. (H) Tandem mass (MS/MS) spectrum of doubly charged Alk14 modified (on K194) R-Ras2 peptide. The b- and y- ions are shown along with the peptide sequence. (I) In-gel fluorescence (with NH2OH treatment) showing that single mutation of four lysine residues at the C-terminus of R-Ras2 did not affect R-Ras2 lysine fatty acylation. (J) Confocal imaging showed that R-Ras2 WT was mainly localized in the plasma membrane in Sirt6 KO MEFs. R-Ras2 WT in Sirt6 WT MEFs as well as R-Ras2 4KR in Sirt6 WT and KO MEFs was localized in both the intracellular vesicles and plasma membrane (n = 5, 5, 5, 6 cells for each sample from left to right, respectively. The images of other cells were shown in Figure 2—figure supplement 4A.DOI:https://dx.doi.org/10.7554/eLife.25158.005Scheme showing the in-gel fluorescence method with Alk14 metabolic labeling to identify R-Ras2 as a lysine fatty acylated protein.FLAG-tagged R-Ras2 protein was enriched from whole cell lysates by FlAG immunoprecipitation. Alk14-labeled R-Ras2 protein was detected by in-gel fluorescence after incorporating BODIPY (B)-azide using click chemistry.DOI:https://dx.doi.org/10.7554/eLife.25158.006Validation of R-Ras2 as the defatty-acylation target of SIRT6. (A) Full gel image of R-Ras2 fatty acylation level with or without NH2OH treatment in Sirt6 WT and KO MEFs. (B) Lysine fatty acylation levels of overexpressed R-Ras2 in Sirt6 WT and KO MEFs. (C) Full gel image of R-Ras2 WT and 4KR fatty acylation levels with or without NH2OH treatment in Sirt6 KO MEFs. (D) In-gel fluorescence showing lysine fatty acylation level of overexpressed R-Ras2 WT and C199S mutant in HEK 293T cells with SIRT6 knockdown. C, control shRNA. S6, SIRT6 shRNA#1.DOI:https://dx.doi.org/10.7554/eLife.25158.007Total ion chromatogram (TIC), extracted ion chromatogram (XIC) and parent MS (MS1) of Ak14 modified R-Ras2 peptide.DOI:https://dx.doi.org/10.7554/eLife.25158.008Lysine fatty acylation targets R-Ras2 to plasma membrane.(A) Confocal imaging showing subcellular localization of GFP-tagged R-Ras2 WT and 4KR in Sirt6 WT and KO MEFs. (B) Subcellular fractionation of R-Ras2 WT and 4KR in HEK 293T cells with palmitic acid or TM3 treatment. GAPDH was used as the marker of cytosol fraction and Na,K-APTase was used as the marker of plasma membrane fraction.DOI:https://dx.doi.org/10.7554/eLife.25158.009 Image collected and cropped by CiteAb from the following open publication (https://elifesciences.org/articles/25158), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: PGAM1/2/4 Antibody [NB100-774] -

Immunocytochemistry/ Immunofluorescence: PGAM1/2/4 Antibody [NB100-774] - Detection of PGAM in KLN-205 cells with antibodies directed to whole PGAM protein or to C-terminal peptide of PGAMA. control conditions (scan parameters in red channel were set to emphasize nucleolar staining with propidium iodide – PI) B. RNase-treated cells. Bar=15 μm. Image collected & cropped by CiteAb from the following publication (https://www.oncotarget.com/lookup/doi/10.18632/oncotarget.4044), licensed under a CC-BY license. Not internally tested by Novus Biologicals.