Journal: Physiological Reports

Article Title: Exercise training reduces ventricular arrhythmias through restoring calcium handling and sympathetic tone in myocardial infarction mice

doi: 10.14814/phy2.13972

Figure Lengend Snippet: (A) The expression of microRNA‐1 (miR‐1) in the LV was elevated in MI‐Sed, and reduced by exercise and β‐blocker treatment. (B) There was no difference in the expression of miR‐133a between the groups ( n = 5/group). (C) There was no difference in expression of protein phosphatase 1 (PP1), while PP2A‐B56‐α (F‐10) expression was significantly decreased in MI‐Sed compared to that in Sham and reversed in MI‐Ex and MI‐βb (D) ( n = 6/group) * P < 0.05, versus Sham; # P < 0.05, versus MI‐Sed.

Article Snippet: The blots were then blocked with the primary antibodies as follows: phospho‐RyR2 (Ser 2814) (A010‐31; Badrilla, Leeds, UK), phospho‐RyR2 (Ser 2808) (A010‐30, Badrilla), RyR2 (PA5‐36121; Thermo Fisher Scientific), SERCA2a (2A7‐A1; Thermo Fisher Scientific), phospho‐Phospholamban (p‐PLN) (S16 + T17; ab62170; Abcam, Cambridge, UK), total Phospholamban (PLN) (ab2865; Abcam), phospho‐PKA (T197; ab75991; Abcam), PKA (ab187515; Abcam), phospho‐CaMKII (T286) (ab32678; Abcam), Oxidized‐CaMKII (Met281/282) (EMD Millipore, Darmstadt, Germany), total CaMKII (ab103840; Abcam), protein phosphatase 2A (PP2A)‐B56‐α (F‐10; sc‐271151; Santa Cruz, Dallas, TX), phospho‐Troponin‐I (p‐TNI) (Ser23/24) (#4004; Cell Signaling Technology, Danvers, MA), total Troponin‐I (TNI) (#4002; Cell Signaling Technology), PP1 (E‐9; sc‐7482; Santa Cruz), and β‐actin (4967s; Cell Signaling Technology).

Techniques: Expressing

Journal: eLife

Article Title: Division of labour between PP2A-B56 isoforms at the centromere and kinetochore

doi: 10.7554/eLife.42619

Figure Lengend Snippet: Schematic representation of the B56α-γ chimeras alongside representative images and line plot analysis of these chimeras in cells arrested in prometaphase with nocodazole. Each graph represents the mean intensities (±SD) from 2 independent experiments. five kinetochore pairs were analysed per cell, for a total of 10 cells per experiment. Intensity is normalized to the maximum signal in each channel in each experiment. Scale bars, 5µm.

Article Snippet: The following antibodies were used for western blotting (at the final concentration indicated): rabbit α-GFP (custom polyclonal, a gift from G. Kops; 1:5000), mouse α-B56γ (clone A-11, sc-374379, Santa Cruz Biotechnology; 1:1000), mouse α-B56α (clone 23, 610615, BD; 1:1000), mouse α-B56δ (clone H-11, sc-271363, Santa Cruz, 1:1000), rabbit α-B56ε (ARP56694-P050, Aviva, 1:1000), mouse α-PPP2CA (clone 1D6, 05–421, Millipore; 1:5000) and rabbit α-PPP2R1A (clone 81G5, #2041, CST; 1:1000), rabbit α-BubR1 (A300-386A, Bethyl; 1:1000), rabbit α-Axin (C76H11, CST; 1:1000), rabbit α-GEF-H1 (155785, Abcam; 1:1000), rabbit α-Kif4a (A301-074A, Bethyl; 1:1000), rabbit α-RepoMan (HPA030049, Sigma; 1:1000) and rabbit α-Actin (A2066, Sigma; 1:5000) and mouse α-alpha-Tubulin (clone B-5-1-2, T5168, Sigma, 1:5000).

Techniques:

Journal: eLife

Article Title: Division of labour between PP2A-B56 isoforms at the centromere and kinetochore

doi: 10.7554/eLife.42619

Figure Lengend Snippet: ( A ) Schematic, representative images and line plot analysis of B56γ and B56γ EPVA localisation in nocodazole-arrested Flp-in HeLa cells. ( B ) Schematic, representative images and line plot analysis of additional B56γ-α chimaeras to define the extra regions in B56α needed to fully switch B56γ from kinetochores to centromeres. Each graph represents the mean intensities (±SD) from at least 2 independent experiments. 5 kinetochore pairs were analysed per cell, for a total of 10 cells per experiment. Intensity is normalized to the maximum signal in each channel in each experiment. Scale bars, 5µm.

Article Snippet: The following antibodies were used for western blotting (at the final concentration indicated): rabbit α-GFP (custom polyclonal, a gift from G. Kops; 1:5000), mouse α-B56γ (clone A-11, sc-374379, Santa Cruz Biotechnology; 1:1000), mouse α-B56α (clone 23, 610615, BD; 1:1000), mouse α-B56δ (clone H-11, sc-271363, Santa Cruz, 1:1000), rabbit α-B56ε (ARP56694-P050, Aviva, 1:1000), mouse α-PPP2CA (clone 1D6, 05–421, Millipore; 1:5000) and rabbit α-PPP2R1A (clone 81G5, #2041, CST; 1:1000), rabbit α-BubR1 (A300-386A, Bethyl; 1:1000), rabbit α-Axin (C76H11, CST; 1:1000), rabbit α-GEF-H1 (155785, Abcam; 1:1000), rabbit α-Kif4a (A301-074A, Bethyl; 1:1000), rabbit α-RepoMan (HPA030049, Sigma; 1:1000) and rabbit α-Actin (A2066, Sigma; 1:5000) and mouse α-alpha-Tubulin (clone B-5-1-2, T5168, Sigma, 1:5000).

Techniques:

Journal: eLife

Article Title: Division of labour between PP2A-B56 isoforms at the centromere and kinetochore

doi: 10.7554/eLife.42619

Figure Lengend Snippet: B56 localisation in B56α-γ chimaeras spanning the entire B56 (Ch1-4: A–C ), a region at the C-terminus (Ch4a-4d: D–F ). ( A, D ) Schematic representation of the B56α-γ chimaeras created. Representative images ( B, E ) and line plot analysis ( C, F ) to show the B56 localisation pattern in each chimaera. ( G ). Alignment of B56 isoforms within region 4d that controls centromere/kinetochore localisation. ( G–H ): Effect of 4 point-mutations within region 4d to convert B56α to the correspond B56γ sequence (B56α TKHG ). Representative images ( H ) and line plot analysis ( I ) of B56α WT or B56α TKHG in cells arrested in prometaphase with nocodazole. Each graph represents the mean intensities (±SD) from 3 independent experiments. 5 kinetochore pairs were analysed per cell, for a total of 10 cells per experiment. Intensity is normalized to the maximum signal in each channel in each experiment. Scale bars, 5µm.

Article Snippet: The following antibodies were used for western blotting (at the final concentration indicated): rabbit α-GFP (custom polyclonal, a gift from G. Kops; 1:5000), mouse α-B56γ (clone A-11, sc-374379, Santa Cruz Biotechnology; 1:1000), mouse α-B56α (clone 23, 610615, BD; 1:1000), mouse α-B56δ (clone H-11, sc-271363, Santa Cruz, 1:1000), rabbit α-B56ε (ARP56694-P050, Aviva, 1:1000), mouse α-PPP2CA (clone 1D6, 05–421, Millipore; 1:5000) and rabbit α-PPP2R1A (clone 81G5, #2041, CST; 1:1000), rabbit α-BubR1 (A300-386A, Bethyl; 1:1000), rabbit α-Axin (C76H11, CST; 1:1000), rabbit α-GEF-H1 (155785, Abcam; 1:1000), rabbit α-Kif4a (A301-074A, Bethyl; 1:1000), rabbit α-RepoMan (HPA030049, Sigma; 1:1000) and rabbit α-Actin (A2066, Sigma; 1:5000) and mouse α-alpha-Tubulin (clone B-5-1-2, T5168, Sigma, 1:5000).

Techniques: Sequencing

Journal: eLife

Article Title: Division of labour between PP2A-B56 isoforms at the centromere and kinetochore

doi: 10.7554/eLife.42619

Figure Lengend Snippet: ( A and B ) Representative images ( A ) and line plots ( B ) of nocodazole-arrested Flp-in HeLa cells expressing YFP-B56 (B56α, B56β, B56γ1, B56γ3, B56δ and B56ε). For line plots, five kinetochore pairs were analysed per cell, for a total of 10 cells per experiment. Graphs represent the mean intensities (±SD) from 3 independent experiments. Intensity is normalized to the maximum signal in each channel in each experiment. ( C–G ) Flp-in HeLa cells treated with siRNA against B56pool, all B56 isoforms except B56α, or all B56 isoforms except B56γ, were analysed for sister chromatid cohesion, SAC strength, Knl1-MELT dephosphorylation and chromosomal alignment. ( C ) Quantification of percentage of chromosome spreads that contain at least one split centromere. Graph represents mean data (+SD) from 3 independent experiments with 50 metaphase spreads quantified per condition per experiment. ( D ) Time-lapse analysis of cells entering mitosis in the presence of nocodazole and 2.5 µM AZ-3146. The graph represents the cumulative data from 50 cells, which is representative of 3 independent experiments. Representative images ( E ) and quantification ( F ) of relative kinetochore intensities of Knl1-pMELT in cells arrested in prometaphase with nocodazole and treated with MG132 for 30 min, followed by 2.5 µM AZ-3146 for the indicated amount of time. 10 cells were quantified per experiment and the graph displays the mean (+SD) of 3 independent experiments. The individual data points for each experiment can be found in the source data. ( G ) Quantification of chromosome misalignment in cells arrested in metaphase with MG-132. At least 100 cells were scored per condition per experiment and graph represents the mean (-SD) of 3 independent experiments. Misalignments were score as mild (1 to 2 misaligned chromosomes), intermediate (3 to 5 misaligned chromosomes), and severe (>5 misaligned chromosomes). Asterisks indicate significance ( : Welch’s t -test, unpaired, : Mann-Whitney test); ns p>0.05, *p≤0.05, **p≤0.01, ****p≤0.0001. Scale bars, 5µm.

Article Snippet: The following antibodies were used for western blotting (at the final concentration indicated): rabbit α-GFP (custom polyclonal, a gift from G. Kops; 1:5000), mouse α-B56γ (clone A-11, sc-374379, Santa Cruz Biotechnology; 1:1000), mouse α-B56α (clone 23, 610615, BD; 1:1000), mouse α-B56δ (clone H-11, sc-271363, Santa Cruz, 1:1000), rabbit α-B56ε (ARP56694-P050, Aviva, 1:1000), mouse α-PPP2CA (clone 1D6, 05–421, Millipore; 1:5000) and rabbit α-PPP2R1A (clone 81G5, #2041, CST; 1:1000), rabbit α-BubR1 (A300-386A, Bethyl; 1:1000), rabbit α-Axin (C76H11, CST; 1:1000), rabbit α-GEF-H1 (155785, Abcam; 1:1000), rabbit α-Kif4a (A301-074A, Bethyl; 1:1000), rabbit α-RepoMan (HPA030049, Sigma; 1:1000) and rabbit α-Actin (A2066, Sigma; 1:5000) and mouse α-alpha-Tubulin (clone B-5-1-2, T5168, Sigma, 1:5000).

Techniques: Expressing, De-Phosphorylation Assay, MANN-WHITNEY

Journal: eLife

Article Title: Division of labour between PP2A-B56 isoforms at the centromere and kinetochore

doi: 10.7554/eLife.42619

Figure Lengend Snippet: Immunoblot of whole cell lysates from nocodazole-arrested HeLa Flp-in cells treated with the indicated siRNA and probed for B56α, B56γ, B56δ, B56ε or tubulin (note B56β was undetectable). Asterix (*) indicate non-specific bands that do not change upon B56 siRNA.

Article Snippet: The following antibodies were used for western blotting (at the final concentration indicated): rabbit α-GFP (custom polyclonal, a gift from G. Kops; 1:5000), mouse α-B56γ (clone A-11, sc-374379, Santa Cruz Biotechnology; 1:1000), mouse α-B56α (clone 23, 610615, BD; 1:1000), mouse α-B56δ (clone H-11, sc-271363, Santa Cruz, 1:1000), rabbit α-B56ε (ARP56694-P050, Aviva, 1:1000), mouse α-PPP2CA (clone 1D6, 05–421, Millipore; 1:5000) and rabbit α-PPP2R1A (clone 81G5, #2041, CST; 1:1000), rabbit α-BubR1 (A300-386A, Bethyl; 1:1000), rabbit α-Axin (C76H11, CST; 1:1000), rabbit α-GEF-H1 (155785, Abcam; 1:1000), rabbit α-Kif4a (A301-074A, Bethyl; 1:1000), rabbit α-RepoMan (HPA030049, Sigma; 1:1000) and rabbit α-Actin (A2066, Sigma; 1:5000) and mouse α-alpha-Tubulin (clone B-5-1-2, T5168, Sigma, 1:5000).

Techniques: Western Blot

Journal: eLife

Article Title: Division of labour between PP2A-B56 isoforms at the centromere and kinetochore

doi: 10.7554/eLife.42619

Figure Lengend Snippet: ( A ) Schematic representation of the strategy used for CRISPR/Cas9 YFP-tagging of B56 isoforms α and γ at the N-terminus. ( B ) Immunoblot of whole cell lysates from nocodazole-arrested HeLa cells with or without YFP-endogenously tagged B56α (enB56α) or B56γ (enB56γ). ( C and D ) Representative images ( C ) and line plots ( D ) of nocodazole-arrested Flp-in HeLa cells expressing endogenously tagged YFP-B56α (enB56α) or YFP-B56γ (enB56γ). For line plots, 5 kinetochore pairs were analysed per cell, for a total of 10 cells per experiment. Graphs represent the mean intensities (±SD) from 3 independent experiments. Intensity is normalized to the maximum signal in each channel in each experiment. Scale bars, 5µm.

Article Snippet: The following antibodies were used for western blotting (at the final concentration indicated): rabbit α-GFP (custom polyclonal, a gift from G. Kops; 1:5000), mouse α-B56γ (clone A-11, sc-374379, Santa Cruz Biotechnology; 1:1000), mouse α-B56α (clone 23, 610615, BD; 1:1000), mouse α-B56δ (clone H-11, sc-271363, Santa Cruz, 1:1000), rabbit α-B56ε (ARP56694-P050, Aviva, 1:1000), mouse α-PPP2CA (clone 1D6, 05–421, Millipore; 1:5000) and rabbit α-PPP2R1A (clone 81G5, #2041, CST; 1:1000), rabbit α-BubR1 (A300-386A, Bethyl; 1:1000), rabbit α-Axin (C76H11, CST; 1:1000), rabbit α-GEF-H1 (155785, Abcam; 1:1000), rabbit α-Kif4a (A301-074A, Bethyl; 1:1000), rabbit α-RepoMan (HPA030049, Sigma; 1:1000) and rabbit α-Actin (A2066, Sigma; 1:5000) and mouse α-alpha-Tubulin (clone B-5-1-2, T5168, Sigma, 1:5000).

Techniques: CRISPR, Western Blot, Expressing

Journal: eLife

Article Title: Division of labour between PP2A-B56 isoforms at the centromere and kinetochore

doi: 10.7554/eLife.42619

Figure Lengend Snippet: ( A-D ) Flp-in HeLa cells treated with siRNA against B56 pool and induced to re-express YFP-B56α or YFP-B56γ were analysed for SAC silencing and chromosomal alignment. Representative images ( A ) and quantification of relative kinetochore intensities ( B ) of Knl1-pMELT in cells arrested in prometaphase with nocodazole and treated with MG132 for 30 min followed by 2.5 μM AZ-3146 for the indicated amount of time. 10 cells were quantified per experiment and graphs represent the mean (+SD) of 4 independent experiments. Scale bars, 5µm. ( C ) Time-lapse analysis of cells entering mitosis in the presence of nocodazole and 2.5 µM AZ-3146. The graph represents the cumulative data from 50 cells, which is representative of 3 independent experiments. ( D ) Quantification of chromosome alignment in cells arrested in metaphase with MG-132. At least 100 cells were scored per condition per experiment and graph represents the mean (-SD) of 3 independent experiments. Misalignments were score as mild (1 to 2 misaligned chromosomes), intermediate (3 to 5 misaligned chromosomes), and severe (>5 misaligned chromosomes). Asterisks indicate significance (Mann-Whitney test); ****p≤0.0001.

Article Snippet: The following antibodies were used for western blotting (at the final concentration indicated): rabbit α-GFP (custom polyclonal, a gift from G. Kops; 1:5000), mouse α-B56γ (clone A-11, sc-374379, Santa Cruz Biotechnology; 1:1000), mouse α-B56α (clone 23, 610615, BD; 1:1000), mouse α-B56δ (clone H-11, sc-271363, Santa Cruz, 1:1000), rabbit α-B56ε (ARP56694-P050, Aviva, 1:1000), mouse α-PPP2CA (clone 1D6, 05–421, Millipore; 1:5000) and rabbit α-PPP2R1A (clone 81G5, #2041, CST; 1:1000), rabbit α-BubR1 (A300-386A, Bethyl; 1:1000), rabbit α-Axin (C76H11, CST; 1:1000), rabbit α-GEF-H1 (155785, Abcam; 1:1000), rabbit α-Kif4a (A301-074A, Bethyl; 1:1000), rabbit α-RepoMan (HPA030049, Sigma; 1:1000) and rabbit α-Actin (A2066, Sigma; 1:5000) and mouse α-alpha-Tubulin (clone B-5-1-2, T5168, Sigma, 1:5000).

Techniques: MANN-WHITNEY

Journal: eLife

Article Title: Division of labour between PP2A-B56 isoforms at the centromere and kinetochore

doi: 10.7554/eLife.42619

Figure Lengend Snippet: ( A ) Immunoblot of whole cell lysates from nocodazole-arrested Flp-in HeLa cells treated with the indicated siRNA expressing endogenous YFP-B56α (enB56α) or exogenous YFP-B56α (ex B56α). Blot was probed for B56α or actin. ( B–D ) Localization of endogenous and exogenous YFP-B56α. Representative images ( B ) and quantification of relative centromere/kinetochore intensity ( C ) of B56α in cells arrested in prometaphase with nocodazole. 10 cells were quantified per experiment and graphs represent the mean (±SD) of 3 independent experiments. Scale bars, 5µm. ( D ) Line plot analysis: five kinetochore pairs were analysed per cell, for a total of 10 cells per experiment. Graphs represent the mean intensities (±SD) from three independent experiments. Intensity is normalized to the maximum signal present in each channel within the endogenous B56α experiment. Asterisks indicate significance (Mann-Whitney test); ****p≤0.0001.

Article Snippet: The following antibodies were used for western blotting (at the final concentration indicated): rabbit α-GFP (custom polyclonal, a gift from G. Kops; 1:5000), mouse α-B56γ (clone A-11, sc-374379, Santa Cruz Biotechnology; 1:1000), mouse α-B56α (clone 23, 610615, BD; 1:1000), mouse α-B56δ (clone H-11, sc-271363, Santa Cruz, 1:1000), rabbit α-B56ε (ARP56694-P050, Aviva, 1:1000), mouse α-PPP2CA (clone 1D6, 05–421, Millipore; 1:5000) and rabbit α-PPP2R1A (clone 81G5, #2041, CST; 1:1000), rabbit α-BubR1 (A300-386A, Bethyl; 1:1000), rabbit α-Axin (C76H11, CST; 1:1000), rabbit α-GEF-H1 (155785, Abcam; 1:1000), rabbit α-Kif4a (A301-074A, Bethyl; 1:1000), rabbit α-RepoMan (HPA030049, Sigma; 1:1000) and rabbit α-Actin (A2066, Sigma; 1:5000) and mouse α-alpha-Tubulin (clone B-5-1-2, T5168, Sigma, 1:5000).

Techniques: Western Blot, Expressing, MANN-WHITNEY

Journal: eLife

Article Title: Division of labour between PP2A-B56 isoforms at the centromere and kinetochore

doi: 10.7554/eLife.42619

Figure Lengend Snippet: ( A-G ) The effect of Sgo1 and/or Sgo2 knockdown on YFP-B56α localisation in Flp-in HeLa cells. Representative images ( A, C, F ) and quantifications ( B, D, G ) of relative kinetochore intensity of B56α in cells arrested in prometaphase with nocodazole after knockdown of Sgo2 ( A, B ), Sgo1 ( C, D ), or Sgo1 +Sgo2 ( F, G ). ( E ) shows line plots of Sgo2 and B56α localisation following Sgo1 knockdown; 5 kinetochore pairs were analysed per cell, for a total of 10 cells per experiment. Graphs represent the mean intensities (±SD) from 3 independent experiments. Intensity is normalized to the maximum signal present in each channel within the endogenous B56α experiment. ( H–M ) Flp-in HeLa cells expressing YFP-B56α or YFP-B56γ were transfected with the CB-Sgo2 ( H–K ) or gChr7 +Cas9 DARPIN ( L, M ) and analysed for B56 recruitment in cells arrested in prometaphase with nocodazole. ( H ), ( L ), and J ). are representative images; I ) and K ) are quantifications of relative centromere/kinetochore intensity of the indicated antigen; and M ) is quantification of intensity of Sgo2 over B56 at the Chr7 locus. For all centromere/kinetochore intensity graphs, each dot represents a cell and 10 cells were quantified per experiment for at least 3 independent experiments. The spread of dots indicates the biological variation between individual cells and the errors bars display the variation between the experimental repeats (displayed as -/+SD of the experimental means). Asterisks indicate significance (Mann-Whitney test); ns p>0.05, *p≤0.05, ****p≤0.0001. Scale bars, 5µm.

Article Snippet: The following antibodies were used for western blotting (at the final concentration indicated): rabbit α-GFP (custom polyclonal, a gift from G. Kops; 1:5000), mouse α-B56γ (clone A-11, sc-374379, Santa Cruz Biotechnology; 1:1000), mouse α-B56α (clone 23, 610615, BD; 1:1000), mouse α-B56δ (clone H-11, sc-271363, Santa Cruz, 1:1000), rabbit α-B56ε (ARP56694-P050, Aviva, 1:1000), mouse α-PPP2CA (clone 1D6, 05–421, Millipore; 1:5000) and rabbit α-PPP2R1A (clone 81G5, #2041, CST; 1:1000), rabbit α-BubR1 (A300-386A, Bethyl; 1:1000), rabbit α-Axin (C76H11, CST; 1:1000), rabbit α-GEF-H1 (155785, Abcam; 1:1000), rabbit α-Kif4a (A301-074A, Bethyl; 1:1000), rabbit α-RepoMan (HPA030049, Sigma; 1:1000) and rabbit α-Actin (A2066, Sigma; 1:5000) and mouse α-alpha-Tubulin (clone B-5-1-2, T5168, Sigma, 1:5000).

Techniques: Expressing, Transfection, MANN-WHITNEY

Journal: eLife

Article Title: Division of labour between PP2A-B56 isoforms at the centromere and kinetochore

doi: 10.7554/eLife.42619

Figure Lengend Snippet: ( A-D ) Flp-in HeLa cells expressing YFP-B56α or YFP-B56γ were transfected with CB-Sgo1 and analysed for B56 recruitment. Representative images ( A and C ) and quantifications ( B and D ) of relative centromere/kinetochore intensity of the indicated antigen in cells arrested in prometaphase with nocodazole. 10 cells were quantified per experiment and graphs represent the mean (±SD) of 4 independent experiments. Asterisks indicate significance (Mann-Whitney test); *****p≤0.0001. Scale bars, 5µm.

Article Snippet: The following antibodies were used for western blotting (at the final concentration indicated): rabbit α-GFP (custom polyclonal, a gift from G. Kops; 1:5000), mouse α-B56γ (clone A-11, sc-374379, Santa Cruz Biotechnology; 1:1000), mouse α-B56α (clone 23, 610615, BD; 1:1000), mouse α-B56δ (clone H-11, sc-271363, Santa Cruz, 1:1000), rabbit α-B56ε (ARP56694-P050, Aviva, 1:1000), mouse α-PPP2CA (clone 1D6, 05–421, Millipore; 1:5000) and rabbit α-PPP2R1A (clone 81G5, #2041, CST; 1:1000), rabbit α-BubR1 (A300-386A, Bethyl; 1:1000), rabbit α-Axin (C76H11, CST; 1:1000), rabbit α-GEF-H1 (155785, Abcam; 1:1000), rabbit α-Kif4a (A301-074A, Bethyl; 1:1000), rabbit α-RepoMan (HPA030049, Sigma; 1:1000) and rabbit α-Actin (A2066, Sigma; 1:5000) and mouse α-alpha-Tubulin (clone B-5-1-2, T5168, Sigma, 1:5000).

Techniques: Expressing, Transfection, MANN-WHITNEY

Journal: eLife

Article Title: Division of labour between PP2A-B56 isoforms at the centromere and kinetochore

doi: 10.7554/eLife.42619

Figure Lengend Snippet: ( A ) Immunoblot of the indicated proteins, containing a LxxIxE motif , following YFP immunoprecipitation from nocodazole-arrested Flp-in HeLa cells expressing YFP-B56α or YFP-B56γ. ( B ) Quantification of the mean normalised intensity (+SD) of the indicated antigens in B56α immunoprecipitates, relative to B56γ immunoprecipitates, from at least 3 independent experiments. Representative images ( C ) and line plot analysis ( D ) of YFP-B56α in Flp-in HeLa cells arrested in nocodazole and treated with the indicated siRNA. Each line plot graph represents the mean intensities (±SD) from 3 independent experiments. 5 kinetochore pairs were analysed per cell, for a total of 10 cells per experiment. Intensity is normalized to the maximum signal in each channel in each experiment. Asterisks indicate significance (Welch’s t -test, unpaired); *p≤0.05, **p≤0.01, ****p≤0.0001. Scale bars, 5µm.

Article Snippet: The following antibodies were used for western blotting (at the final concentration indicated): rabbit α-GFP (custom polyclonal, a gift from G. Kops; 1:5000), mouse α-B56γ (clone A-11, sc-374379, Santa Cruz Biotechnology; 1:1000), mouse α-B56α (clone 23, 610615, BD; 1:1000), mouse α-B56δ (clone H-11, sc-271363, Santa Cruz, 1:1000), rabbit α-B56ε (ARP56694-P050, Aviva, 1:1000), mouse α-PPP2CA (clone 1D6, 05–421, Millipore; 1:5000) and rabbit α-PPP2R1A (clone 81G5, #2041, CST; 1:1000), rabbit α-BubR1 (A300-386A, Bethyl; 1:1000), rabbit α-Axin (C76H11, CST; 1:1000), rabbit α-GEF-H1 (155785, Abcam; 1:1000), rabbit α-Kif4a (A301-074A, Bethyl; 1:1000), rabbit α-RepoMan (HPA030049, Sigma; 1:1000) and rabbit α-Actin (A2066, Sigma; 1:5000) and mouse α-alpha-Tubulin (clone B-5-1-2, T5168, Sigma, 1:5000).

Techniques: Western Blot, Immunoprecipitation, Expressing

Journal: eLife

Article Title: Division of labour between PP2A-B56 isoforms at the centromere and kinetochore

doi: 10.7554/eLife.42619

Figure Lengend Snippet: ( A ) Immunoblot of the PP2A subunits following YFP immunoprecipitation from nocodazole-arrested Flp-in HeLa cells expressing YFP-B56α WT or YFP-B56α TKHG. ( B ) Flp-in HeLa cells treated with siRNA against B56 pool and induced to re-express indicted antigens were subjected to time-lapse analysis to determine mitotic timing. The graph represents the cumulative data from 50 cells, which is representative of 3 independent experiments.

Article Snippet: The following antibodies were used for western blotting (at the final concentration indicated): rabbit α-GFP (custom polyclonal, a gift from G. Kops; 1:5000), mouse α-B56γ (clone A-11, sc-374379, Santa Cruz Biotechnology; 1:1000), mouse α-B56α (clone 23, 610615, BD; 1:1000), mouse α-B56δ (clone H-11, sc-271363, Santa Cruz, 1:1000), rabbit α-B56ε (ARP56694-P050, Aviva, 1:1000), mouse α-PPP2CA (clone 1D6, 05–421, Millipore; 1:5000) and rabbit α-PPP2R1A (clone 81G5, #2041, CST; 1:1000), rabbit α-BubR1 (A300-386A, Bethyl; 1:1000), rabbit α-Axin (C76H11, CST; 1:1000), rabbit α-GEF-H1 (155785, Abcam; 1:1000), rabbit α-Kif4a (A301-074A, Bethyl; 1:1000), rabbit α-RepoMan (HPA030049, Sigma; 1:1000) and rabbit α-Actin (A2066, Sigma; 1:5000) and mouse α-alpha-Tubulin (clone B-5-1-2, T5168, Sigma, 1:5000).

Techniques: Western Blot, Immunoprecipitation, Expressing

Journal: eLife

Article Title: Division of labour between PP2A-B56 isoforms at the centromere and kinetochore

doi: 10.7554/eLife.42619

Figure Lengend Snippet: ( A-D ) Flp-in HeLa cells expressing either YFP-B56α WT or TKHG were transfected with the CB-Sgo2 and analysed for B56 recruitment ( A, B ) or gChr7 +dCas9 DARPIN to assess YFP-B56α:Sgo2 co-localisation ( C, D ). Representative images ( A, C ) and quantification of relative kinetochore intensity ( B ) or intensity of Sgo2 over B56α at the Chr7 locus ( D ). For the intensity graphs in B ) and D ), each dot represents a cell and 10 cells were quantified per experiment from at least 3 independent experiments. The spread of dots indicates the biological variation between individual cells and the errors bars display the variation between experimental repeats (displayed as -/+SD of the experimental means). ( E ) Immunoblot of the indicated antigens following immunoprecipitation of YFP from nocodazole-arrested Flp-in HeLa cells expressing YFP- B56γ, YFP-B56α WT or YFP-B56α-TKHG. ( F ) Quantification of the mean normalised intensity (+SD) of indicated antigens in B56α WT or B56α TKHG immunoprecipitates, relative to B56γ, from at least 4 experiments. ( G ) Crystal structure of PP2A-B56γ [accession code 2NPP ] with annotation to indicate the regions that specify localisation to centromeres or kinetochores (see for details of the additional centromere/kinetochore specification regions). Note, the structure is meant only as a guide because the critical regions may be present within B56α, which has not been structurally solved. The B56α WT values are also used in some of the points plotted in . Asterisks indicate significance (Mann-Whitney test, except : Welch’s t -test, unpaired); ns p>0.05, *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001. Scale bars, 5µm.

Article Snippet: The following antibodies were used for western blotting (at the final concentration indicated): rabbit α-GFP (custom polyclonal, a gift from G. Kops; 1:5000), mouse α-B56γ (clone A-11, sc-374379, Santa Cruz Biotechnology; 1:1000), mouse α-B56α (clone 23, 610615, BD; 1:1000), mouse α-B56δ (clone H-11, sc-271363, Santa Cruz, 1:1000), rabbit α-B56ε (ARP56694-P050, Aviva, 1:1000), mouse α-PPP2CA (clone 1D6, 05–421, Millipore; 1:5000) and rabbit α-PPP2R1A (clone 81G5, #2041, CST; 1:1000), rabbit α-BubR1 (A300-386A, Bethyl; 1:1000), rabbit α-Axin (C76H11, CST; 1:1000), rabbit α-GEF-H1 (155785, Abcam; 1:1000), rabbit α-Kif4a (A301-074A, Bethyl; 1:1000), rabbit α-RepoMan (HPA030049, Sigma; 1:1000) and rabbit α-Actin (A2066, Sigma; 1:5000) and mouse α-alpha-Tubulin (clone B-5-1-2, T5168, Sigma, 1:5000).

Techniques: Expressing, Transfection, Western Blot, Immunoprecipitation, MANN-WHITNEY

Journal: eLife

Article Title: Division of labour between PP2A-B56 isoforms at the centromere and kinetochore

doi: 10.7554/eLife.42619

Figure Lengend Snippet: Immunoblot of LxxIxE containing proteins (GEF-H1, BubR1 and RepoMan) following YFP immunoprecipitation from nocodazole-arrested Flp-in HeLa cells expressing YFP-B56α or YFP-B56γ and subjected to control or Sgo2 siRNA, as indicated.

Article Snippet: The following antibodies were used for western blotting (at the final concentration indicated): rabbit α-GFP (custom polyclonal, a gift from G. Kops; 1:5000), mouse α-B56γ (clone A-11, sc-374379, Santa Cruz Biotechnology; 1:1000), mouse α-B56α (clone 23, 610615, BD; 1:1000), mouse α-B56δ (clone H-11, sc-271363, Santa Cruz, 1:1000), rabbit α-B56ε (ARP56694-P050, Aviva, 1:1000), mouse α-PPP2CA (clone 1D6, 05–421, Millipore; 1:5000) and rabbit α-PPP2R1A (clone 81G5, #2041, CST; 1:1000), rabbit α-BubR1 (A300-386A, Bethyl; 1:1000), rabbit α-Axin (C76H11, CST; 1:1000), rabbit α-GEF-H1 (155785, Abcam; 1:1000), rabbit α-Kif4a (A301-074A, Bethyl; 1:1000), rabbit α-RepoMan (HPA030049, Sigma; 1:1000) and rabbit α-Actin (A2066, Sigma; 1:5000) and mouse α-alpha-Tubulin (clone B-5-1-2, T5168, Sigma, 1:5000).

Techniques: Western Blot, Immunoprecipitation, Expressing

Journal: eLife

Article Title: Division of labour between PP2A-B56 isoforms at the centromere and kinetochore

doi: 10.7554/eLife.42619

Figure Lengend Snippet:

Article Snippet: The following antibodies were used for western blotting (at the final concentration indicated): rabbit α-GFP (custom polyclonal, a gift from G. Kops; 1:5000), mouse α-B56γ (clone A-11, sc-374379, Santa Cruz Biotechnology; 1:1000), mouse α-B56α (clone 23, 610615, BD; 1:1000), mouse α-B56δ (clone H-11, sc-271363, Santa Cruz, 1:1000), rabbit α-B56ε (ARP56694-P050, Aviva, 1:1000), mouse α-PPP2CA (clone 1D6, 05–421, Millipore; 1:5000) and rabbit α-PPP2R1A (clone 81G5, #2041, CST; 1:1000), rabbit α-BubR1 (A300-386A, Bethyl; 1:1000), rabbit α-Axin (C76H11, CST; 1:1000), rabbit α-GEF-H1 (155785, Abcam; 1:1000), rabbit α-Kif4a (A301-074A, Bethyl; 1:1000), rabbit α-RepoMan (HPA030049, Sigma; 1:1000) and rabbit α-Actin (A2066, Sigma; 1:5000) and mouse α-alpha-Tubulin (clone B-5-1-2, T5168, Sigma, 1:5000).

Techniques: Recombinant, Clone Assay, Mutagenesis, Plasmid Preparation, Sequencing, Knock-In, Modification, Magnetic Beads, Software, Western Blot, Microscopy

Journal: Molecular Cancer Therapeutics

Article Title: Small-Molecule–Mediated Stabilization of PP2A Modulates the Homologous Recombination Pathway and Potentiates DNA Damage-Induced Cell Death

doi: 10.1158/1535-7163.MCT-21-0880

Figure Lengend Snippet: Combination of SMAP-061 and PARPi synergistically engages the DDR pathway and prevents DNA repair by specifically targeting and downregulating RAD51. A, Western blot analysis evaluating dose-dependency effects of SMAP-061, olaparib, or combination on DDR protein expression and activity. Sensor, transducer, and effector proteins (from schematic) were analyzed in OV81, PEO-1, and PEO-C42. B, RAD51 foci imaging comparing DMSO with 20 μmol/L SMAP-061, 100 μmol/L olaparib or combination of SMAP+olaparib in OV81 cells treated after 24 hours of incubation (left) and their respective quantification bar graph (right). Data presented as the mean ± SEM ( n = 3; unpaired Student t tests, comparing each group with each other, **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). C, OV81, PEO-1, and PEO-C4.2 cell lines were exposed to increasing doses of either SMAP-061 alone (blue) or in combination with 80 μmol/L of 766 (a biologically inactive analogue of SMAP-061; red), and cell viability was measured at 48 hours by MTT analysis, to generate an EC 50 curve. Data are presented as the mean ± SD ( n = 3). D, Rescue experiments were performed on OV81, PEO-1, and PEO-C42 cells treated with DMSO, SMAP-061, 766, or SMAP + 766. Rescue profile analysis of DDR (BRCA1/2 and RAD51) protein expression as well as recovery in cell death markers (cleaved Parp and cleaved Caspase-3) and DNA damage signaling capacity (γH2Ax and p-RPA) after 12 hours of treatment are analyzed by Western blotting. Values underneath RAD51 blot represent the fold change of protein expression to its DMSO control sample, after Vinculin normalization. E, Quantification of RAD51 protein expression, with data presented as the mean ± SEM ( n = 3; unpaired Student t tests, comparing each treatment group relative with its respective DMSO; *, P < 0.05). F, Calyculin-A (PP2A Catalytic subunit inhibitor) rescue experiments were performed using Western blotting (and respective quantification of RAD51 in G ). H, MTT techniques to assess cellular viability and an EC 50 value shift for OV81, PEO-1, and PEO-C4.2. 5 nmol/L of Calyculin-A was preincubated for 1 hour, followed by SMAP-061 treatment. Data presented as the mean ± SD ( n = 3; unpaired Student t tests, comparing each treatment group relative with each other, *, P < 0.05; ***, P < 0.001). I, CRISPR Cas9 KO of 2 specific B-subunits (PPP2R2A and PPP2R5A) rescue experiments were performed using Western blotting looking at HR sensors (γH2Ax and p-RPA), RAD51 effector, and apoptotic markers (cleaved PARP and cleaved Caspase3). Representative quantification is located under each blot and its respective lane. Each target was normalized to Vinculin. p/t-RPA ratio was calculated before normalization to Vinculin. J, Clonogenic assay of OV81 CTRL CRISPR, PPP2R2A KO #1, and PPP2R5A KO #2. Cells were treated with DMSO or 10 μmol/L SMAP-061 for 2 weeks ( n = 3; Left). Quantification the clonogenic assay comparing OV81 control (CTRL CRISPR), PPP2R2A KO #1, and PPP2R5A KO #2. Data presented as the mean ± SD ( n = 3; unpaired Student t tests, comparing each treatment group with its own DMSO control, *, P < 0.05; Right).

Article Snippet: Guide RNAs to PPP2R5A (B56α) and PPP2R2A (B55α) sequences were obtained from GenScript that were deposited and licensed by the Feng Zheng laboratory at the Broad Institute.

Techniques: Western Blot, Expressing, Activity Assay, Imaging, Incubation, Control, CRISPR, Clonogenic Assay

Journal: Emerging Microbes & Infections

Article Title: To be or not to be phosphorylated: understanding the role of Ebola virus nucleoprotein in the dynamic interplay with the transcriptional activator VP30 and the host phosphatase PP2A-B56

doi: 10.1080/22221751.2024.2447612

Figure Lengend Snippet: Transcription activation requires a close spatial and orientational interplay between NP, PP2A-B56α, and VP30. (A) EBOV NP mutants carrying mutations at the PP2A-B56α (orange) and VP30 (green) binding interfaces that were utilized for the following experiments. (B) Transcription and replication competent virus-like particle (trVLP) assay with a tetracistronic minigenome, reporter gene activity in producer cells (p0). 72 hours post transfection (hpt), luciferase activity was measured reflecting EBOV-specific transcription. NPwt set to 100%. Mean of individual experiments, ns: not statistically significant. (C) Reporter gene activity in infected target cells (p1). Generated trVLPs from (B) supernatants were collected and concentrated via ultracentrifugation to infect naïve p1 cells. Luciferase activity was measured 72 hpt reflecting primary transcription. NPwt set to 100%. Mean of individual experiments. (D) Expression of NP and VP40 in p0 cell lysates and incorporation into trVLPs analysed by WB analysis. Samples generated as described in panels B and C. (E–F) Co-immunoprecipitation of NP mutants with B56α (E) or VP30 (F) in HEK293 cells using anti-flag antibody covered magnetic beads. Flag-tagged NP is precipitated together with potential binding partners that are subsequently detected through WB. On the right side, quantification from individual experiments. NPwt set to 100%. (G) VP30 phosphorylation in the presence of NP upon recombinant expression in HeLa cells. VP30AA was included as positive control for dephosphorylated VP30 (lane 11 ). On the right side, quantification from individual experiments. NPwt set to 100%.

Article Snippet: We could recently determine the underlying mechanism of how EBOV and also Marburg virus VP30 are dephosphorylated by PP2A: the regulatory subunit B56α of PP2A as well as its substrate VP30 directly bind to NP, that thereby acts as a scaffold protein bringing both proteins in close contact, a mechanism that is conserved among filoviruses [ , ].

Techniques: Activation Assay, Binding Assay, Virus, Activity Assay, Transfection, Luciferase, Infection, Generated, Expressing, Immunoprecipitation, Magnetic Beads, Phospho-proteomics, Recombinant, Positive Control

Journal: Emerging Microbes & Infections

Article Title: To be or not to be phosphorylated: understanding the role of Ebola virus nucleoprotein in the dynamic interplay with the transcriptional activator VP30 and the host phosphatase PP2A-B56

doi: 10.1080/22221751.2024.2447612

Figure Lengend Snippet: Rescue and characterization of recEBOV-NP mutants with an increased distance between the binding sites of PP2A-B56α and VP30. (A) Successfully rescued recEBOV as indicated. Bottom, scheme of NP with the naturally emerged compensatory mutation NP-T603I in the recEBOV-NP58aa context. (B) Growth kinetics of the in panel A introduced recEBOVs. VeroE6 cells were infected with the indicated MOI and supernatant was collected at 0-, 1-, 2-, 3-, 4-, and 7-dpi. Viral titres were determined by TCID 50 /ml and a mean from three independent experiments is shown. (C) Viral inclusion body formation in HuH7 cells infected with the respective viruses (MOI 0.3) at 24 hpi. NP and VP40 were stained with specific antibodies, and nuclei are stained with DAPI. (D) Protein expression in lysates and supernatants at 24 hpi from the samples generated in (C). (E) Viral protein incorporation in equal amounts of infectious virus particles (PFU). 2 × 10 5 PFU were concentrated by ultracentrifugation for each of the respected recEBOVs and analysed by WB. (F) Viral genome copies in equal amounts of infectious virus particles (PFU). Mean of 5 individual experiments is shown. C t values (as 45– C t ) are shown based on EBOV-specific RT-qPCR. (G) Ratio of incorporated viral proteins in recEBOV purified from HuH7 cells determined by MS/MS. Mean and standard deviation determined from four independent experiments. For individual measurements, see supplement S4. (H) Cryo-electron microscopy images of recEBOVs purified from HuH7 cells.

Article Snippet: We could recently determine the underlying mechanism of how EBOV and also Marburg virus VP30 are dephosphorylated by PP2A: the regulatory subunit B56α of PP2A as well as its substrate VP30 directly bind to NP, that thereby acts as a scaffold protein bringing both proteins in close contact, a mechanism that is conserved among filoviruses [ , ].

Techniques: Binding Assay, Mutagenesis, Infection, Staining, Expressing, Generated, Virus, Quantitative RT-PCR, Purification, Tandem Mass Spectroscopy, Standard Deviation, Cryo-Electron Microscopy

Journal: Emerging Microbes & Infections

Article Title: To be or not to be phosphorylated: understanding the role of Ebola virus nucleoprotein in the dynamic interplay with the transcriptional activator VP30 and the host phosphatase PP2A-B56

doi: 10.1080/22221751.2024.2447612

Figure Lengend Snippet: Model of NP and VP30 phosphorylation contributing to an efficient EBOV life cycle. NP phosphorylation state differs between virions and in cells. EBOV NP is phosphorylated at position T603 in virions, upon infection of target cells; NP-T603 requires dephosphorylation to support efficient viral transcription and replication. At a later time point during the viral replication cycle, NP-T603 is re-phosphorylated and finally packaged into newly formed virions. NP serves as a key player in the tight interplay of VP30-dependent transcription initiation by facilitating VP30 dephosphosphorylation through PP2A-B56α.

Article Snippet: We could recently determine the underlying mechanism of how EBOV and also Marburg virus VP30 are dephosphorylated by PP2A: the regulatory subunit B56α of PP2A as well as its substrate VP30 directly bind to NP, that thereby acts as a scaffold protein bringing both proteins in close contact, a mechanism that is conserved among filoviruses [ , ].

Techniques: Phospho-proteomics, Infection, De-Phosphorylation Assay