mouse anti methylated pp2ac antibody  (Santa Cruz Biotechnology)

Journal: Frontiers in Cell and Developmental Biology

Article Title: A Novel Role of PP2A Methylation in the Regulation of Tight Junction Assembly and Integrity

doi: 10.3389/fcell.2022.911279

Figure Lengend Snippet: Expression of the L309Δ mutant in MDCK cells alters PP2A distribution and complex formation with TJ proteins and Par3. (A) Polarized MDCK monolayers were analyzed by confocal microscopy for the distribution of WTC and L309Δ using anti-HA antibodies. Representative apical x-y and transversal x-z sections are shown; the arrow indicates the monolayer thickness. (B) Representative distribution of PP2A-Bα in MDCK-WTC and MDCK-L309Δ cells 20 h after a Ca 2+ switch. (C) Western blot analysis of HA-PP2Ac and PP2A-Bα in total lysates and NP40-detergent-insoluble fractions prepared from MDCK-WTC and MDCK-L309Δ cells cultured in NC medium. Sodium potassium pump (Na,K-ATPase), membrane marker. (D) Western blot analyses of ZO-1 and occludin in total lysates and HA-immunoprecipitates (IP) prepared from MDCK cells transfected with WTC, L309Δ, or empty vector (EV). (E) Western blot analyses of endogenous Par3 and aPKCζ in total lysates and HA immunoprecipitates (IP) prepared from WTC-, L309Δ- or EV-, transfected MDCK cells. (F) Western blot analyses of myc-Par3 and HA-PP2Ac in total lysates and corresponding myc immunoprecipitates (IP) prepared from MDCK-Par3 cells stably expressing myc-Par3, that were transfected with either WTC, L309Δ, or EV. IgG, immunoglobulin. For (A–F) , similar results were obtained in three separate experiments. Scale bars = 10 μm.

Article Snippet: Antibodies used in this study included: Mouse (clone 16B12, Covance) and rabbit (clone C29F4, Cell Signaling Technology) anti-HA; mouse anti-Myc (clone 9B11, Cell Signaling Technology); mouse anti-methyl PP2Ac (clone 2A10, Merck Millipore); mouse anti-demethyl-PP2Ac (clone 1D6; Merck Millipore); mouse anti-PP2Ac (#610556, BD Transduction); mouse anti-LCMT1 clone 4A4 (Merck Millipore); mouse anti-PME-1 ( ); rabbit ( ) and mouse (clone 2G9, Merck Millipore) anti-Bα; rabbit anti-Na + /K + ATPase (#3010, Cell Signaling Technology); mouse anti-occludin (clone OC-3F10; Thermo Fisher Scientific); mouse anti-ZO-1 (clone ZO1-1A12, Thermo Fisher Scientific); rabbit anti-Par3 (#07-330, Merck-Millipore); rabbit anti-aPKCζ (sc-216, Santa Cruz Biotechnology); mouse (clone C4, Merck Millipore) and rabbit (#AAN01, Cytoskeleton Inc.) anti-actin; mouse anti-α-tubulin (clone DM1A; Sigma).

Techniques: Expressing, Mutagenesis, Confocal Microscopy, Western Blot, Cell Culture, Membrane, Marker, Transfection, Plasmid Preparation, Stable Transfection

Journal: Frontiers in Cell and Developmental Biology

Article Title: A Novel Role of PP2A Methylation in the Regulation of Tight Junction Assembly and Integrity

doi: 10.3389/fcell.2022.911279

Figure Lengend Snippet: Overexpression of LCMT1 and PME1 in MDCK cells affects the initial redistribution of ZO-1, occludin and Par3 to cell-cell contacts during Ca 2+ -induced TJ reassembly. (A) Total lysates from MDCK-LCMT1 and control MDCK cells were analyzed by Western blot (left) for HA-LCMT1, LCMT1, PP2A-Bα, and total/methylated/demethylated PP2Ac levels. Relative expression levels (right) of PP2A-Bα and methylated PP2Ac were quantified in these cells (mean ± SEM; n = 3; Student’s t -test; *** p < 0.001). (B) Representative immunoblots of myc-PME1, PME1, PP2A-Bα, and total/methylated/demethylated PP2Ac levels in total lysates from MDCK-PME1 and control MDCK cells (left). The relative levels of demethylated PP2Ac (mean ± SEM; n = 3; Student’s t -test; *** p < 0.001) were quantified in these cells (right). (C) Distribution of ZO-1, occludin and Par3 in control MDCK and MDCK-LCMT1 cells 3 h after a Ca 2+ switch. A subset of MDCK-LCMT1 cells was incubated with 100 nM OA (+OA) during the switch. (D) Distribution of ZO-1, occludin and Par3 in control MDCK and MDCK-PME1 cells switched for 2 h from LC to NC medium. For (C–D) , representative images from three separate experiments are shown. Scale bars = 10 μm.

Article Snippet: Antibodies used in this study included: Mouse (clone 16B12, Covance) and rabbit (clone C29F4, Cell Signaling Technology) anti-HA; mouse anti-Myc (clone 9B11, Cell Signaling Technology); mouse anti-methyl PP2Ac (clone 2A10, Merck Millipore); mouse anti-demethyl-PP2Ac (clone 1D6; Merck Millipore); mouse anti-PP2Ac (#610556, BD Transduction); mouse anti-LCMT1 clone 4A4 (Merck Millipore); mouse anti-PME-1 ( ); rabbit ( ) and mouse (clone 2G9, Merck Millipore) anti-Bα; rabbit anti-Na + /K + ATPase (#3010, Cell Signaling Technology); mouse anti-occludin (clone OC-3F10; Thermo Fisher Scientific); mouse anti-ZO-1 (clone ZO1-1A12, Thermo Fisher Scientific); rabbit anti-Par3 (#07-330, Merck-Millipore); rabbit anti-aPKCζ (sc-216, Santa Cruz Biotechnology); mouse (clone C4, Merck Millipore) and rabbit (#AAN01, Cytoskeleton Inc.) anti-actin; mouse anti-α-tubulin (clone DM1A; Sigma).

Techniques: Over Expression, Control, Western Blot, Methylation, Expressing, Incubation

Journal: Frontiers in Cell and Developmental Biology

Article Title: A Novel Role of PP2A Methylation in the Regulation of Tight Junction Assembly and Integrity

doi: 10.3389/fcell.2022.911279

Figure Lengend Snippet: Endogenous PME1-mediated PP2A demethylation is enhanced during early Ca 2+ -induced TJ formation in MDCK cells. (A) Endogenous demethylated PP2Ac levels were analyzed by immunoblotting (top) and quantified (bottom) in MDCK cells switched for 0, 1 and 4 h from LC to NC medium. Data (mean ± SD) were appraised using one-way ANOVA (F = 168.8; p < 0.0001) with post hoc Dunnett’s multiple comparisons test. **** p < 0.0001. (B) Representative distribution of ZO-1, occludin and Par3 in MDCK cells incubated for 4 h with either 20 μM AMZ-30 or vehicle during a Ca 2+ switch. Similar results were obtained in three separate experiments. Scale bar = 10 μm. (C) Relative levels of demethylated PP2Ac were quantified in these cells (mean ± SEM; n = 3; Student’s t -test; **** p < 0.0001).

Article Snippet: Antibodies used in this study included: Mouse (clone 16B12, Covance) and rabbit (clone C29F4, Cell Signaling Technology) anti-HA; mouse anti-Myc (clone 9B11, Cell Signaling Technology); mouse anti-methyl PP2Ac (clone 2A10, Merck Millipore); mouse anti-demethyl-PP2Ac (clone 1D6; Merck Millipore); mouse anti-PP2Ac (#610556, BD Transduction); mouse anti-LCMT1 clone 4A4 (Merck Millipore); mouse anti-PME-1 ( ); rabbit ( ) and mouse (clone 2G9, Merck Millipore) anti-Bα; rabbit anti-Na + /K + ATPase (#3010, Cell Signaling Technology); mouse anti-occludin (clone OC-3F10; Thermo Fisher Scientific); mouse anti-ZO-1 (clone ZO1-1A12, Thermo Fisher Scientific); rabbit anti-Par3 (#07-330, Merck-Millipore); rabbit anti-aPKCζ (sc-216, Santa Cruz Biotechnology); mouse (clone C4, Merck Millipore) and rabbit (#AAN01, Cytoskeleton Inc.) anti-actin; mouse anti-α-tubulin (clone DM1A; Sigma).

Techniques: Western Blot, Incubation

Journal: Frontiers in Cell and Developmental Biology

Article Title: A Novel Role of PP2A Methylation in the Regulation of Tight Junction Assembly and Integrity

doi: 10.3389/fcell.2022.911279

Figure Lengend Snippet: Hcy, HTL and SAM metabolites modulate PP2A methylation state and TJ assembly. (A) Total and demethylated PP2Ac were analyzed by Western blot (top) in total lysates from MDCK cells incubated for 24 h in NC medium in the presence of either 100 μM Hcy, 100 μM HTL, 100 μM SAM, or vehicle. Demethylated PP2Ac levels were quantified in these cells (bottom). Data (mean ± SEM; n = 3) were appraised using one-way ANOVA (F = 150.6; p < 0.0001) with post hoc Dunnett’s test. **** p < 0.0001, *** p < 0.001, versus vehicle-treated controls. (B) Distribution of ZO-1, occludin and Par3 in these cells. Arrows indicate examples of ruffled TJs. (C) Quantification of TJ ruffling in SAM-treated MDCK cells (mean ± SEM; Student’s t -test; **** p < 0.0001). (D) TER values were measured in cells from (B) . Data (mean ± SEM; n = 6 Transwells) were analyzed using one-way ANOVA (F = 42.12; p < 0.0001) with post hoc Dunnett’s multiple comparisons test. **** p < 0.0001, *** p = 0.001, * p < 0.05, versus vehicle-treated control cells. (E) Distribution of ZO-1 and Par3 in MDCK cells switched for 2 h from LC to NC medium in the presence of either 200 μM HTL or vehicle. (F) Distribution of ZO-1 and Par3 in MDCK cells switched for 4 h from LC to NC medium containing either 200 μM Hcy or HTL, together with 20 μM AMZ-30 or vehicle. (G) Distribution of ZO-1 in MDCK-LCMT1 cells switched for 4 h from LC to NC medium in the presence of either 200 μM SAM or vehicle. For (B,E–G) , representative confocal images are shown; similar results were obtained in three separate experiments. Scale bars = 10 μm.

Article Snippet: Antibodies used in this study included: Mouse (clone 16B12, Covance) and rabbit (clone C29F4, Cell Signaling Technology) anti-HA; mouse anti-Myc (clone 9B11, Cell Signaling Technology); mouse anti-methyl PP2Ac (clone 2A10, Merck Millipore); mouse anti-demethyl-PP2Ac (clone 1D6; Merck Millipore); mouse anti-PP2Ac (#610556, BD Transduction); mouse anti-LCMT1 clone 4A4 (Merck Millipore); mouse anti-PME-1 ( ); rabbit ( ) and mouse (clone 2G9, Merck Millipore) anti-Bα; rabbit anti-Na + /K + ATPase (#3010, Cell Signaling Technology); mouse anti-occludin (clone OC-3F10; Thermo Fisher Scientific); mouse anti-ZO-1 (clone ZO1-1A12, Thermo Fisher Scientific); rabbit anti-Par3 (#07-330, Merck-Millipore); rabbit anti-aPKCζ (sc-216, Santa Cruz Biotechnology); mouse (clone C4, Merck Millipore) and rabbit (#AAN01, Cytoskeleton Inc.) anti-actin; mouse anti-α-tubulin (clone DM1A; Sigma).

Techniques: Methylation, Western Blot, Incubation, Control