co2 agarose beads  (Thermo Fisher)

Journal: BMC Cell Biology

Article Title: Acetylation of C-terminal lysines modulates protein turnover and stability of Connexin-32

doi: 10.1186/s12860-018-0173-0

Figure Lengend Snippet: HDAC6 inhibition reduces turnover and increases acetylation of Cx32. a TSA and ( b ) TubA increase expression level of Cx32. Western blots of N2a cell lysates treated with the TSA (concentrations as indicated) ( a ) or 20 μM TubA for 6 h ( b ) were probed with anti-Cx32, actin ( a ) or tubulin ( b ) was used as a loading control. c TubA treatment decreases turnover of Cx32 (bottom panel) compared to untreated samples (top panel). Protein turnover of 35-S-Cx32 was determined by phosphorimaging at 0, 1, 2, 4 h post-chase (shown). Blots were subsequently probed with anti-Myc as a loading control. d Relative levels of 35-S-Cx32 remaining after 1.5 h post-chase (in subsequent experiments) was quantified by densitometry of phosphorimages, normalized to Myc, and used to calculate half-life, as described (see Methods). ( N = 4 independent experiments; student’s T-test* p < 0.05 as compared to DMSO control). e TubA increases expression levels of total Cx32 and acetylated Cx32. Cx32 immunoprecipitates of N2a cell lysates expressing Cx32-Myc/6xHis treated with TubA or vehicle alone were processed for Western blots analysis and probed with Cx32 and AcK antibodies

Article Snippet: For His-tagged Cx32, Hispur-Co2+ coated agarose beads (Pierce) were added to the cleared supernatant and incubated as per manufacturer’s suggestions.

Techniques: Inhibition, Expressing, Western Blot, Control

Journal: BMC Cell Biology

Article Title: Acetylation of C-terminal lysines modulates protein turnover and stability of Connexin-32

doi: 10.1186/s12860-018-0173-0

Figure Lengend Snippet: HDAC6 inhibition results in the accumulation of both cytoplasmic and cell surface Cx32. a TubA treatment stabilizes Cx32 levels as determined by increases in levels of biotinylated Cx32 at 1.5 h after labeling. N2a cell were transfected with Cx32-Myc/6xHis and treated with TubA or vehicle for 3 h prior to biotinylation of cell surface proteins. Following cell surface biotinylation, cultures were harvested and processed for total lysate (top) and biotinylated Cx32-Myc/6xHis (bottom) by Western blot, as described in Methods. b Densitometric quantification of Western blots shows a significant reduction in biotinylated Cx32/total Cx32 at T0 in TubA-treated samples as compared to vehicle-treated cells. c A significant increase in biotinylated Cx32/total Cx32 remained at T1.5 compared to vehicle-treated ( N = 3 independent experiments, Student’s T-test * p < 0.05). d Immunostaining and confocal microscopy cell surface labeling of Cx32 confirmed TubA treatment increases Cx32 accumulation in cell surface and cytoplasmic compartments (see also Additional file : Fig. S1). Images shown were obtained at 63X magnification and scale bars = 25 μm. Arrows indicate points of cell-cell contact

Article Snippet: For His-tagged Cx32, Hispur-Co2+ coated agarose beads (Pierce) were added to the cleared supernatant and incubated as per manufacturer’s suggestions.

Techniques: Inhibition, Labeling, Transfection, Western Blot, Immunostaining, Confocal Microscopy

Journal: BMC Cell Biology

Article Title: Acetylation of C-terminal lysines modulates protein turnover and stability of Connexin-32

doi: 10.1186/s12860-018-0173-0

Figure Lengend Snippet: C-terminal lysines are required for HDAC6 inhibitor-induced accumulation of Cx32. a Mutation of C-terminal (bottom), but not cytoplasmic loop (top), lysines prevented Cx32 accumulation in response to TubA treatment. N2a cells transfected with either Cx32-Myc/6xHis or site-specific Cx32-Myc/6xHis K➔R acetylation-deficient mutants were treated with DMSO or TubA and processed for Western blot. Blots were probed for Myc-tag to detect Cx32, with beta-tubulin as a loading control. (Note that vertical lines between samples in figure indicate use of sister blots, run simultaneously.) ( b ) N2a cells were transfected with either Cx32-Myc/6xHis (WT), acetylation-deficient multi-site lysine to arginine mutant of all five cytoplasmic C-terminal lysines (5R), or acetylation-mimetic multi-site lysine to glutamine mutant of the five C-terminal lysines (5Q). WT, 5R, 5Q expressing cultures were treated with TubA or vehicle and assessed for Cx32 expression as in ( a ). c WT, 5R, 5Q expressing cultures were treated with TubA or vehicle and assessed for acetylation with anti-AcK. Cultures were immunoprecipitated with affinity purified rabbit-anti-Cx32 (E. Hertzberg) and probed with mouse monoclonal anti-Cx32 (7C6.7C; E. Hertzberg) or anti-AcK. Basal levels of acetylation were lower, as predicted, in untreated 5R and increased in 5Q relative to total Cx32. Treatment with TubA increased WT and 5Q acetylation, while 5R levels remained largely unchanged

Article Snippet: For His-tagged Cx32, Hispur-Co2+ coated agarose beads (Pierce) were added to the cleared supernatant and incubated as per manufacturer’s suggestions.

Techniques: Mutagenesis, Transfection, Western Blot, Control, Expressing, Immunoprecipitation, Affinity Purification

Journal: BMC Cell Biology

Article Title: Acetylation of C-terminal lysines modulates protein turnover and stability of Connexin-32

doi: 10.1186/s12860-018-0173-0

Figure Lengend Snippet: C-terminal lysines modulate Cx32 ubiquitination. a Ubiquitination levels of 5R, and 5Q Cx32, relative to WT, was assessed by probing Western blots of Co2+ affinity-purified Cx32-Myc/6xHis with antibodies against pan-ubiquitin and Myc-tag. Note that monoubiquitin has a molecular mass of 8.5 kDa and thus the bands shown here could represent polyubiquitinated and multi-mono-ubiquitinated Cx32. b Densitometric quantification of Ub-Cx32 as a percentage of WT Ub-Cx32 is shown here. Relative levels of Ub-Cx32 were 47% and 15% for 5R and 5Q, respectively, versus WT ( N = 3 experiments, Student’s T-test: * p < 0.05)

Article Snippet: For His-tagged Cx32, Hispur-Co2+ coated agarose beads (Pierce) were added to the cleared supernatant and incubated as per manufacturer’s suggestions.

Techniques: Ubiquitin Proteomics, Western Blot, Affinity Purification

Journal: BMC Cell Biology

Article Title: Acetylation of C-terminal lysines modulates protein turnover and stability of Connexin-32

doi: 10.1186/s12860-018-0173-0

Figure Lengend Snippet: C-terminal lysines modulate Cx32 turnover. a Cells expressing Myc/6xHis tagged WTCx32, 5RCx32, or 5QCx32 were pulsed with 35-S-Met/Cys for 5 min and lysed (0) or chased 1.5 h in complete medium (1.5). Cx32-Myc/His was isolated by affinity purification and processed for Western blotting. 35S-Met/Cys incorporation into newly synthesized Cx32-Myc/His was detected by phosphorimaging and the blots were subsequently probed with anti-Myc antibody to detect Cx32-Myc/His. b Densitometric quantification of 35-S Cx32 remaining after 1.5 h. 35-S was quantified and normalized to total Cx32-Myc amount (* p < 0.05, Student’s T-test, N = 3)

Article Snippet: For His-tagged Cx32, Hispur-Co2+ coated agarose beads (Pierce) were added to the cleared supernatant and incubated as per manufacturer’s suggestions.

Techniques: Expressing, Isolation, Affinity Purification, Western Blot, Synthesized

Journal: BMC Cell Biology

Article Title: Acetylation of C-terminal lysines modulates protein turnover and stability of Connexin-32

doi: 10.1186/s12860-018-0173-0

Figure Lengend Snippet: C-terminal lysine mutants form functional gap junctions. a Dual whole-cell recordings showed no significant differences in junctional conductance (Gj) between WT, 5R, or 5Q cell pairs. Mean Junctional conductances for pIRES2EGFP (I), WT, 5R, and 5Q Cx32 are plotted. b Cells in experiments plotted here were transfected with 3-fold more DNA than in A; no influence on Gj activity is observed. c Representative average normalized Gj-Vj relations with 5R (left) and 5Q (right) are compared with Cx32WT channels. The average normalized instantaneous (open triangles) and steady-state (filled squares) junctional conductance (Gj) at each Vj were calculated from current traces and fit as described in the Materials and Methods. Solid lines represent fits to Cx32-5R or Cx32-5Q; broken lines represent fits to data for Cx32WT homotypic cell-cell channels

Article Snippet: For His-tagged Cx32, Hispur-Co2+ coated agarose beads (Pierce) were added to the cleared supernatant and incubated as per manufacturer’s suggestions.

Techniques: Functional Assay, Transfection, Activity Assay

Journal: BMC Cell Biology

Article Title: Acetylation of C-terminal lysines modulates protein turnover and stability of Connexin-32

doi: 10.1186/s12860-018-0173-0

Figure Lengend Snippet: Cellular proliferation is regulated by Cx32 acetylation. BrdU incorporation by N2a cells expressing the pCx32-IRESEGFP WT, 5R, or 5Q constructs were quantified by immunofluorescent staining for Cx32 and BrdU, as described in methods. Following overnight incubation with medium containing BrdU, cells were fixed, labeled and scored for Cx32 expression and BrdU incorporation. The percentage of BrdU-positive and Cx32 co-expressing cells were normalized to the total number of Cx32-expressing cells for each construct. a The percentage of BrdU positive cells in 5R samples were significantly reduced compared to WT (* p < 0.0026). b No differences in the percentage of Cx32 positive cells per total DAPI positive nuclei (cell number) were noted. For each construct, three to four fields were imaged and quantified for each replicate coverslip per experiment using the MultiWavelength Cell Scoring app in Metamorph, as described in the Methods. ( N = 3 experiments for each construct). c Representative images of BrdU incorporation by N2a cells expressing the pCx32-IRESEGFP WT, 5R, or 5Q constructs, labeled and processed as described above

Article Snippet: For His-tagged Cx32, Hispur-Co2+ coated agarose beads (Pierce) were added to the cleared supernatant and incubated as per manufacturer’s suggestions.

Techniques: BrdU Incorporation Assay, Expressing, Construct, Staining, Incubation, Labeling