Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: RAB23 facilitates clathrin-coated nascent vesicle formation at the plasma membrane and modulates cell signaling

doi: 10.1007/s00018-025-05694-w

Figure Lengend Snippet:

Article Snippet: GFP-Trap based co-immunoprecipitation was performed using GFP-Trap A agarose beads (iST GFP-Trap Test Kit, Kit for AP-MS sample preparation of GFP-fusion proteins, Chromotek; gtak-iST-8,).

Techniques: Marker, Recombinant, Lysis, Blocking Assay, Protease Inhibitor, Transfection, Bicinchoninic Acid Protein Assay, Derivative Assay, Plasmid Preparation, Software, Imaging

Journal: bioRxiv

Article Title: The endoplasmic reticulum stress sensor IRE1 regulates collagen secretion through the enforcement of the proteostasis factor P4HB/PDIA1 contributing to liver damage and fibrosis

doi: 10.1101/2023.05.02.538835

Figure Lengend Snippet: (a) Real time qPCR of P4HB mRNA levels in U20S (single clone) (n=5), Hepa1-6 (single clone) (n=5), and Huh7 (pool) (n=5) IRE1null CRISPR-Cas9 (KO, red) and their corresponding CRISPR-Cas9 control (WT, blue). using two-tailed unpaired Student’s t -tests. Mean ± SEM are shown. *P < 0.05; **P < 0.01; ****P < 0.0001.(b) Immunoblots of P4HB with indicative loading controls in U20S Hepa1-6 and Huh7 IRE1null CRISPR-Cas9 (KO) and their corresponding CRISPR-Cas9 control (WT). (c) Representative histograms of GFP-collagen fluorescence in the presence or absence of chloroquine (autophagy inhibitor, 50 μm) and MG132 (proteasome inhibitor, 50 μm) for 16 h. Quantification of relative change in the median fluorescence intensity with respect to the untreated. Results are mean ± SEM of 3 independent experiments. Statistical analysis was performed using two-tailed unpaired Student’s t -tests. Mean ± SEM are shown (d) GFP-collagen cell’s nucleus was stained with the Dapi and GFP-collagen punta’s were quantified using ImageJ software of 3 independent experiments (n=574 CCT-collagen cells and n=639 KO-collagen cells). Statistical analysis was performed using two-tailed unpaired Student’s t -tests. Mean ± SEM are shown. (e) GFP-collagen cell’s nucleus was stained with the Dapi, ER with KDEL and GFP-collagen punta’s were quantified using ImageJ software of 2 independent experiments (n=372 CCT-collagen cells and n=363 KO-collagen cells). Statistical analysis was performed using two-tailed unpaired Student’s t-tests. Mean ± SEM are shown. (f) Filter-trap assay for collagen and GFP antibody. (g) Represents the CLEM of the WT-GFP-collagen and KO-GFP-collagen with given scale bar.

Article Snippet: In brief, to immunoprecipitated GFP-tagged Collagen, 600 ug of protein extracts were incubated with mouse anti-GFP antibody (SCBT) for 2 h at 4 °C, followed by the incubation with 15 ul of SureBeadsTM Protein G Magnetic Beads for 1 h at 4°C.

Techniques: CRISPR, Control, Two Tailed Test, Western Blot, Fluorescence, Staining, Software, TRAP Assay

Journal: bioRxiv

Article Title: The endoplasmic reticulum stress sensor IRE1 regulates collagen secretion through the enforcement of the proteostasis factor P4HB/PDIA1 contributing to liver damage and fibrosis

doi: 10.1101/2023.05.02.538835

Figure Lengend Snippet: (a) (a) Represents the sorting strategy for selecting cells based on normalized GFP signal. (b) Regular splicing assay for Xbp1 mRNA for WT, KO, WT-GFP-collagen, and KO-GFP-collagen after 4 and 8 h of Tm treatment. (c) Representative fluorescence of Bodipy red ER-tracker indicating the extent of stained red stained ER. Data was analyzed using FlowJoTMv10 software. Quantification represents the median fluorescence intensity. Results are mean ± SEM of 3 independent experiments. Statistical analysis was performed by using one-way ANOVA with Tukey’s multiple comparison test. (d) Representative TEM images of CCT, KO, CCT-GFP-collagen, and KO-GFP-collagen U2OS cells showing the morphology and extent of ER (scale bar 1 μm). Quantification represents the area of ER (μm 2 ) including above Q1 and below Q3. Data represents mean ±SEM from one experiment (n=23 CCT cells, n=55 KO cells, n=41 CCT- collagen cells and n=65 KO-collagen cells). Statistical analysis was performed by using one-way ANOVA with Tukey’s multiple comparison test.

Article Snippet: In brief, to immunoprecipitated GFP-tagged Collagen, 600 ug of protein extracts were incubated with mouse anti-GFP antibody (SCBT) for 2 h at 4 °C, followed by the incubation with 15 ul of SureBeadsTM Protein G Magnetic Beads for 1 h at 4°C.

Techniques: Splicing Assay, Fluorescence, Staining, Software, Comparison

Journal: bioRxiv

Article Title: The endoplasmic reticulum stress sensor IRE1 regulates collagen secretion through the enforcement of the proteostasis factor P4HB/PDIA1 contributing to liver damage and fibrosis

doi: 10.1101/2023.05.02.538835

Figure Lengend Snippet: (a) Representative histograms of GFP-collagen fluorescence in presence of indicating ascorbate concentrations for 24 h. Quantification of median fluorescence intensity were measured using FlowJo TM v10 software. Results are mean ± SEM of 4 independent experiments. Statistical analysis was performed by using two-way ANOVA with Tukey’s multiple comparison test. (b) Immunoblotting of WT-GFP-collagen and KO-GFP-collagen cells post 48 h transfection with P4HB in presence or absence of ascorbate after 24 h incubation for indicative antibodies and approximate molecular weight. (c) Immunofluorescence on WT, KO, WT-GFP-collagen, and KO-GFP-collagen U2OS cell post 48 h transfection with V5-P4HB in presence or absence of ascorbate after 24 h incubation probed for GFP-collagen, KDEL, V5 and Dapi. (d) Co-immunoprecipitation of P4HB with WT-GFP-collagen or KO-GFP-collagen U2OS cells. Total extracts and IP were analyzed by Western blot using specific antibodies for P4HB and GFP. WT U2OS cells were used as control of immunoprecipitation.

Article Snippet: In brief, to immunoprecipitated GFP-tagged Collagen, 600 ug of protein extracts were incubated with mouse anti-GFP antibody (SCBT) for 2 h at 4 °C, followed by the incubation with 15 ul of SureBeadsTM Protein G Magnetic Beads for 1 h at 4°C.

Techniques: Fluorescence, Software, Comparison, Western Blot, Transfection, Incubation, Molecular Weight, Immunofluorescence, Immunoprecipitation, Control

Journal: bioRxiv

Article Title: The endoplasmic reticulum stress sensor IRE1 regulates collagen secretion through the enforcement of the proteostasis factor P4HB/PDIA1 contributing to liver damage and fibrosis

doi: 10.1101/2023.05.02.538835

Figure Lengend Snippet: (a) Representative half offset histograms of GFP-collagen fluorescence post 48 h transfection with P4HB, ERp57 and ERp72 with presence or absence of ascorbate after 24 h incubation. Quantification of median fluorescence intensity was measured using FlowJoTMv10 software. Results are mean ± SEM of 3 independent experiments. Statistical analysis was performed by using two-way ANOVA with Tukey’s multiple comparison test. (b) Immunoblotting of WT-GFP-collagen and KO-GFP-collagen cells post 48 h transfection with P4HB in presence or absence of ascorbate after 24 h incubation for indicative antibodies and approximate molecular weight. (c) Filter-trap assay performed on the WT, KO, WT-GFP-collagen, and KO-GFP-collagen U2OS cell post 48 h transfection with V5-P4HB in presence or absence of ascorbate after 24 h incubation against collagen antibody. (d) Immunofluorescence on WT, KO, WT-GFP-collagen, and KO-GFP-collagen U2OS cell post 48 h transfection with V5-P4HB in presence or absence of ascorbate after 24 h incubation probed for GFP-collagen, KDEL, V5 and Dapi.

Article Snippet: In brief, to immunoprecipitated GFP-tagged Collagen, 600 ug of protein extracts were incubated with mouse anti-GFP antibody (SCBT) for 2 h at 4 °C, followed by the incubation with 15 ul of SureBeadsTM Protein G Magnetic Beads for 1 h at 4°C.

Techniques: Fluorescence, Transfection, Incubation, Software, Comparison, Western Blot, Molecular Weight, TRAP Assay, Immunofluorescence

Journal: Nature Communications

Article Title: A plant RNA virus inhibits NPR1 sumoylation and subverts NPR1-mediated plant immunity

doi: 10.1038/s41467-023-39254-2

Figure Lengend Snippet: a Growth of serially diluted yeast cells that were transformed with the indicated plasmids on selective medium. b Confocal microscopic photographs of N. benthamiana epidermal cells that were infiltrated by agrobacteria harboring the indicated plasmids at 2 dpi. Scale bar, 50 μm. The experiment was independently repeated three times with similar results. c Co-IP assay to test the interaction between NIb and NPR1 or SUMO3. FLAG-4×Myc or YFP-tagged proteins were expressed in N. benthamiana leaves by agroinfiltration, immunoprecipitated with GFP-trap agarose at 2 dpi, and detected with anti-GFP N-terminal (anti-GFP-N) or anti-Myc antibodies, respectively. Asterisks indicate nonspecific bands. The experiment was independently repeated twice with similar results. d In vitro binding assay with purified proteins from E. coli . GST or GST-NIb was used as matrix-bound bait to bind TrxA-6×His or TrxA-6×His-NPR1. The asterisk indicates degraded NPR1. The experiment was independently repeated twice with similar results. e Schematic representation of NPR1. Numbers represent amino acid positions of domain boundaries. Ser11/Ser15, Cys82, Cys156, Cys216, His334, and SIM3 are also indicated. f Confocal microscopic photographs of N. benthamiana epidermal cells expressing NIb-YN and YC-tagged NPR1-truncated mutants or sim3 at 2 dpi. Scale bar, 50 μm. The experiment was independently repeated three times with similar results. g Growth of serially diluted yeast cells that were transformed with BD-NIb and AD-tagged NPR1-truncated mutants or sim3 on selective medium.

Article Snippet: The resulting supernatant was added to 30 μL of Chromotek GFP-Trap (Proteintech, Wuhan, Hubei, China) or anti-FLAG M2 affinity gel (Sigma‒Aldrich, Shanghai, China) and incubated for 3 h at 4 °C with gentle rotation.

Techniques: Transformation Assay, Co-Immunoprecipitation Assay, Immunoprecipitation, In Vitro, Binding Assay, Purification, Expressing

Journal: Nature Communications

Article Title: A plant RNA virus inhibits NPR1 sumoylation and subverts NPR1-mediated plant immunity

doi: 10.1038/s41467-023-39254-2

Figure Lengend Snippet: a Evaluation of the interaction between NIb and NPR1 mutants by Y2H. The experiment was performed as shown in Fig. except that the incubation time was reduced due to the fast growth rate of yeast cells transformed with BD-NIb and S11/15D-AD. b Box and whisker plot with individual data points showing the nuclear YFP intensity in N. benthamiana epidermal cells expressing NIb-YC and YN-tagged NPR1 or its mutants. The data are the mean of the nuclear signal intensity of 5 micrographs. The whisker indicates minimum and maximum, and the box indicates the 25th and 75th percentiles (edges of the box), and median (center line). The original figures are available in the Figshare repository under [10.6084/m9.figshare.23243918]. c Phenotypes of 3-week-old WT, 35S::sim3 | S11/15A-GFP ( 35S::sim3|S11/15A ), 35S::sim3|S11/15D-GFP ( 35S::sim3|S11/15D ), and 35S::S11/15D-GFP ( 35S::S11/15D ) seedlings under steady-state conditions. d Phenotypes of mock or TuMV-infected WT and transgenic plants at 18 dpi. e Bar plot showing the ratio of virus-infected to total leaf area of WT and transgenic plants at 18 dpi ( n = 5). f Immunoblot analysis of the phosphorylation of transiently expressed NPR1 or its mutants in the presence of GUS, NIb or NIb sim2 by Phos-tag. Equal amounts of GFP-Trap agarose-enriched NPR1 or its mutants were analyzed by anti-NPR1 antibodies or treated with Phos-tag. Numbers represent relative Phos-tag intensities that are corrected according to the total protein amount. FLAG-4×Myc-SUMO3 and RFP-tagged GUS, NIb or NIb sim2 were detected with anti-Myc and anti-mRFP antibodies, respectively. The experiment was independently repeated twice with similar results. Data are presented as mean values ± SD. Statistical analyses were performed using two-tailed Student’s t test. Source data are provided as a Source Data file.

Article Snippet: The resulting supernatant was added to 30 μL of Chromotek GFP-Trap (Proteintech, Wuhan, Hubei, China) or anti-FLAG M2 affinity gel (Sigma‒Aldrich, Shanghai, China) and incubated for 3 h at 4 °C with gentle rotation.

Techniques: Incubation, Transformation Assay, Whisker Assay, Expressing, Infection, Transgenic Assay, Western Blot, Two Tailed Test

Journal: Nature Communications

Article Title: A plant RNA virus inhibits NPR1 sumoylation and subverts NPR1-mediated plant immunity

doi: 10.1038/s41467-023-39254-2

Figure Lengend Snippet: a Multiple alignment of partial amino acid sequences of NPR1 homologs of different plants. GenBank accession numbers are indicated at the front, and SIM3 is highlighted by a black box with a conserved aa pattern on the bottom. b Evaluation of the interaction between GmNPR1 and GmSUMO homologs by Y2H assay. c Evaluation of the interaction between SMV NIb ( SMV NIb) and GmNPR1 and Gmsim3 by split-luciferase assay. d Analysis of the sumoylation of GmNPR1 by Co-IP assay. Proteins were expressed in N. benthamiana leaves by agroinfiltration, analyzed by western blotting (left panel) or immunoprecipitated with anti-FLAG M2 affinity gel and then detected with anti-Myc and anti-GFP-N antibodies (right panel) at 2 dpi. Sumoylated GmNPR1 is indicated. To avoid overexposure, samples that were detected with anti-Myc antibodies were diluted 20 times for SDS‒PAGE. The experiment was independently repeated twice with similar results. e Western blot analysis of the influence of SMV NIb on GmNPR1 sumoylation. Proteins were expressed in N. benthamiana leaves and analyzed at 2 dpi by Western blotting with anti-Myc, anti-GFP-N, and anti-mRFP polyclonal antibodies (left panel). GmSUMO5 was immunoprecipitated by anti-FLAG M2 affinity gel and then analyzed by anti-Myc or anti-GFP antibodies. Nonmodified and posttranslationally modified GmNPR1 are indicated by an arrow and square bracket, respectively. Similar results were observed in three independent experiments.

Article Snippet: The resulting supernatant was added to 30 μL of Chromotek GFP-Trap (Proteintech, Wuhan, Hubei, China) or anti-FLAG M2 affinity gel (Sigma‒Aldrich, Shanghai, China) and incubated for 3 h at 4 °C with gentle rotation.

Techniques: Y2H Assay, Luciferase, Co-Immunoprecipitation Assay, Western Blot, Immunoprecipitation, Modification

Journal: Nature Communications

Article Title: The spliceosome-associated protein CWC15 promotes miRNA biogenesis in Arabidopsis

doi: 10.1038/s41467-024-46676-z

Figure Lengend Snippet: a The interactions with CWC15 with DCL1, HYL1, SE and DDL analyzed by BiFC. Paired cYFP- and nYFP-fusion proteins were co-expressed in leaves of N. benthamiana . Green color indicates the BiFC signal detected by a confocal microscopy at 48 h after infiltration. Scale bar = 10 μm. Three independent experiments yielded consistent results. b Co-IP between MYC-CWC15 and HYL1−2HA. HYL1-2HA and GFP-2HA were co-expressed with MYC-CWC15 in leaves of N. benthamiana , respectively. IPs were performed using anti-MYC antibodies. HYL1-2HA, GFP-2HA and MYC-CWC15 were detected by western blot. Two independent experiments yielded consistent results. c Co-IP between MYC-CWC15 and SE-2H. SE-2HA and GFP-2HA were co-expressed with MYC-CWC15 in leaves of N. benthamiana , respectively. IPs were performed using anti-MYC antibodies. SE-2HA, GFP-2HA and MYC-CWC15 were detected by western blot. Two independent experiments yielded consistent results. d Co-IP between CWC15 and SE or HYL1 in pCWC15::GFP-CWC15/ amiR CWC15 transgenic plants. Co-IP was performed using anti-GFP antibodies. GFP-CWC15, SE and HYL1 were detected by western blot. Two independent experiments yielded consistent results. Source data are provided as a Source Data file.

Article Snippet: For the interaction of CWC15 and SE or HYL1 in Arabidopsis, IP was performed with ChromoTek GFP-Trap® Agarose in pCWC15::eGFP-CWC15 transgenic plant.

Techniques: Confocal Microscopy, Co-Immunoprecipitation Assay, Western Blot, Transgenic Assay

Journal: Nature Communications

Article Title: The spliceosome-associated protein CWC15 promotes miRNA biogenesis in Arabidopsis

doi: 10.1038/s41467-024-46676-z

Figure Lengend Snippet: a The protein interactions analyzed by BiFC. Paired cYFP- and nYFP-fusion proteins were co-expressed in tobacco leaves. Green color indicates the BiFC signal detected by a confocal microscopy at 48 h after infiltration. Scale bar = 10 μm. Three independent experiments yielded consistent results. b The association of CWC15 with PAG1 and PAB1 detected by Co-IP. PAG1−2HA, PAB1-2HA and GFP-2HA were co-expressed with MYC-CWC15 in leaves of N. benthamiana , respectively. IPs were performed using anti-MYC antibodies. PAG1-2HA, PAB1-2HA, GFP-2HA and MYC-CWC15 were detected by western blot. Two independent experiments yielded consistent results. c Co-IP between CWC15 and PAG1 in plants containing the p35S::2HA-CWC15 and pPAG1::gPAG1-FM transgenes. 2HA-CWC15 and PAG1-FM were detected by western blot. Numbers on top of the pictures indicate plants used: 1, 2HA-CWC15/gPAG1-FM; 2, Col-0; 3, gPAG1-FM. Two independent experiments yielded consistent results. d The enrichment of SE in the PAG1 complex with or without MG132 treatment. IP was performed using anti-FLAG antibodies. SE and PAG1-FM were detected by western blot using anti-SE and anti-FLAG, respectively. Numbers on top of the pictures indicate plants used: 1, Col-0; 2, gPAG1-FM, 3, amiR CWC15 /gPAG1-FM. The enriched SE protein compared with IPed PAG1-FM in amiR CWC15 was normalized to that of Col-0, which was assigned a value of 1. Two independent experiments yielded consistent results. Source data are provided as a Source Data file.

Article Snippet: For the interaction of CWC15 and SE or HYL1 in Arabidopsis, IP was performed with ChromoTek GFP-Trap® Agarose in pCWC15::eGFP-CWC15 transgenic plant.

Techniques: Confocal Microscopy, Co-Immunoprecipitation Assay, Western Blot

Journal: Nature Communications

Article Title: The spliceosome-associated protein CWC15 promotes miRNA biogenesis in Arabidopsis

doi: 10.1038/s41467-024-46676-z

Figure Lengend Snippet: a Interaction between CWC15 and PRP4KA detected by BiFC. Paired cYFP- and nYFP-fusion proteins were co-expressed in leaves of N. benthamiana . Green color indicates the BiFC signal detected by a confocal microscopy at 48 h after infiltration. Scale bar = 10 μm. Three independent experiments yielded consistent results. b , c Co-IP between CWC15 and PRP4KA. 2HA-CWC15 was co-expressed with FM-PRP4KA or GFP−2FLAG in N. benthamiana leaves, respectively. IPs were performed using anti-FLAG antibodies or and anti-HA antibodies. 2HA-CWC15, FM-PRP4KA and GFP-2FLAG were detected by western blot. Two independent experiments yielded consistent results. d Phosphorylation status of endogenous SE in amiR CWC15 and Col-0. SE was detected by SDS-PAGE and phosphor-tag gel in parallel using anti-SE antibodies. e Phosphorylation levels of SE in amiR CWC15 relative to Col. Phosphorylated SE proteins were compared with total SE proteins (set as 1) in Col and amiR CWC15 , respectively. Error bars indicate SD from two biological replicates ( n = 2) and data are presented as mean values +/- SD. Asterisks indicate significantly reduced phosphorylation compared to Col-0 plants based on P- value (two-tailed unpaired t -test; P < 0.01): 0.002787. f The proposed action model of CWC15 in miRNA biogenesis. CWC15 binds the MIR promoters to facilitate the recruitment of Pol II, which may depend on the function of CDC5. CWC15 interacts with SE and HYL1 to enhance pri-miRNA processing. Moreover, CWC15 interacts with PRP4KA and the 20 S proteosome to promote phosphorylation-dependent SE degradation. Source data are provided as a Source Data file.

Article Snippet: For the interaction of CWC15 and SE or HYL1 in Arabidopsis, IP was performed with ChromoTek GFP-Trap® Agarose in pCWC15::eGFP-CWC15 transgenic plant.

Techniques: Confocal Microscopy, Co-Immunoprecipitation Assay, Western Blot, SDS Page, Two Tailed Test

Journal: Journal of Cell Science

Article Title: PP2A­-B55γ counteracts Cdk1 and regulates proper spindle orientation through the cortical dynein adaptor NuMA

doi: 10.1242/jcs.243857

Figure Lengend Snippet: PP2A-B55γ dephosphorylates pT2055. (A) Schematic of B55γ construct with FLAG (FL) and AcGFP tags at the C-terminus (B55γ–AcGFP). (B) Characterization of HeLa Kyoto cell line stably expressing B55γ–AcGFP using western blot analysis. β-actin is shown as a loading control. (C) Co-immunoprecipitation (IP) by GFP-Trap from lysates of nocodazole-arrested mitotic HeLa cells stably expressing B55γ–AcGFP. Resulting blots were probed for NuMA, PPP2CA, PPP2R1B, p150Glued, and GFP, as indicated. IN, input (1% of total); IP, 50% of the total immunoprecipitate; FT, flow-through. For GFP detection in the IP fraction, only 10% of the IP fraction was loaded. Note, B55γ interacts with NuMA, but not with the dynein-interacting dynactin subunit p150Glued. (D,E) Metaphase HeLa Kyoto control cells (D), or cells stably expressing B55γ–AcGFP (E) were fixed and stained for NuMA (red) and DNA (blue). Arrows indicate cortical localization, box indicates example region used for quantification. Note enrichment in cortical NuMA in cells expressing B55γ–AcGFP. Data are mean±s.d. cortical NuMA levels of 12 cells. P=0.0061 between control and B55γ–AcGFP-expressing cells (two-tailed Student's t-test). (F,G) Control metaphase HeLa cells (F), or cells stably expressing B55γ–AcGFP (G) fixed and stained for pT2055 (gray) and DNA (blue). Circle indicates example region used for quantification. Note significantly diminished levels of spindle pole pT2055 in cells expressing B55γ–AcGFP. Data are mean±s.d. spindle pole pT2055 levels of 20 cells. P<0.0001 between control and B55γ–AcGFP-expressing cells (two-tailed Student's t-test). (H) Schematic representation of the in vitro dephosphorylation assay whereby 6His–NuMA(1876–2115) is phosphorylated by Cdk1/cyclinB and detected by anti-pT2055 antibody. This phosphorylated substrate was utilized in the dephosphorylation reaction using IP fraction from B55γ–AcGFP-expressing cells. (I) Detection of phosphorylated 6His–NuMA(1876–2115) with anti-pT2055 antibody. Please note that 6His–NuMA(1876–2115) is unstable, therefore two species are observed in the Coomassie-stained gel and western blots. (J) Dephosphorylation reaction with Cdk1/cyclinB-phosphorylated 6His–NuMA(1876–2115) and the B55γ–AcGFP IP fraction at a different times, as indicated. Note that in this and other dephosphorylation experiments, values below the pT2055 western blot represent the band intensity, normalized to the intensity value from the control sample. HIS, blot using anti-His-tag antibody. (K) Schematic representation of the in vitro dephosphorylation assay as mentioned above, however, the dephosphorylation reaction is performed by either incubating the B55γ–AcGFP IP fraction with anti-GFP antibody (GFP-Ab) to mask AcGFP–B55γ, or with a B55γ–AcGFP IP fraction from cells depleted of PPP2CA catalytic subunit. (L) Dephosphorylation reaction with the B55γ–AcGFP IP that was incubated with GFP-Ab before the dephosphorylation reaction. (M) Dephosphorylation reaction with the B55γ–AcGFP IP fraction from the cells depleted for PPP2CA by RNAi. Please note a substantial decrease in dephosphorylation potential was observed for B55γ–AcGFP IP fractions that were either incubated with GFP-Ab (L) or from cells that were depleted for PPP2CA (M). **P<0.01, ***P<0.0001 (two-tailed Student's t-test).

Article Snippet: For immunoprecipitation, 3 mg of cell lysates procured from the nocodazole (100 nM)-arrested mitotic HeLa Kyoto cells expressing either AcGFP-tagged or GFP-tagged proteins were incubated with 30 μl GFP-Trap agarose beads (Chromotek, ACT-CM-GFA0050) in lysis buffer [50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 25 mM sodium fluoride, 0.1 mM sodium orthovanadate (Sigma-Aldrich, S6508), 0.1 mM PMSF (Calbiochem, 7110), 0.2% Triton-X100, 0.3% NP-40, 100 nM okadaic acid and Complete EDTA-free protease inhibitor (Merck, 539134)] for 4 h at 4°C.

Techniques: Construct, Stable Transfection, Expressing, Western Blot, Immunoprecipitation, Staining, Two Tailed Test, In Vitro, De-Phosphorylation Assay, Incubation

Journal: Journal of Cell Science

Article Title: PP2A­-B55γ counteracts Cdk1 and regulates proper spindle orientation through the cortical dynein adaptor NuMA

doi: 10.1242/jcs.243857

Figure Lengend Snippet: B55γ-PP2A is necessary for NuMA cortical localization in cells mimicking anaphase. (A) Experimental protocol for chemically induced anaphase onset by acute Cdk1 inactivation using RO-3306 treatment in the presence of the kinesin-5 inhibitor STLC for cells stably co-expressing AcGFP–NuMA and mCherry–H2B, or AcGFP–NuMA4KR>A and mCherry–H2B. (B–D) Time-lapse confocal images of HeLa cells stably co-expressing AcGFP–NuMA (green) and mCherry–H2B (magenta) that were either transfected with control siRNAs and treated with STLC and RO-3306 (B, Control), transfected with B55γ siRNAs and treated with STLC and RO-3306 (C), or co-transfected with PPP2CA, B55γ and PPP2R1B siRNAs then treated with STLC and RO-3306 (D). Note the absence of cortical GFP signal in cells that were either transfected with B55γ siRNAs or with PPP2CA, B55γ and PPP2R1B siRNAs, in contrast to control cells. Arrows indicate cortical localization of NuMA. (E) Time-lapse confocal images of HeLa cells stably co-expressing AcGFP–NuMA4KR>A (green) and mCherry–H2B (magenta) and were treated with STLC and RO-3306. Note the substantially weaker cortical GFP signal in these cells upon RO-3306 treatment. (F) Quantification of GFP cortical enrichment in cells as described for B–E. Note significantly reduced cortical signal in cells that were either transfected with B55γ siRNAs, PPP2CA, B55γ and PPP2R1B siRNAs, or expressing AcGFP–NuMA4KR>A upon treatment with RO-3306 for 10 min as indicated. Data show mean±s.d. for more than 10 cells, with individual data points plotted. ***P<0.0001 (two-tailed Student's t-test). A.U., arbitrary units.

Article Snippet: For immunoprecipitation, 3 mg of cell lysates procured from the nocodazole (100 nM)-arrested mitotic HeLa Kyoto cells expressing either AcGFP-tagged or GFP-tagged proteins were incubated with 30 μl GFP-Trap agarose beads (Chromotek, ACT-CM-GFA0050) in lysis buffer [50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 25 mM sodium fluoride, 0.1 mM sodium orthovanadate (Sigma-Aldrich, S6508), 0.1 mM PMSF (Calbiochem, 7110), 0.2% Triton-X100, 0.3% NP-40, 100 nM okadaic acid and Complete EDTA-free protease inhibitor (Merck, 539134)] for 4 h at 4°C.

Techniques: Stable Transfection, Expressing, Transfection, Two Tailed Test

Journal: Journal of Cell Science

Article Title: PP2A­-B55γ counteracts Cdk1 and regulates proper spindle orientation through the cortical dynein adaptor NuMA

doi: 10.1242/jcs.243857

Figure Lengend Snippet: Polybasic residues in the vicinity of T2055 are necessary for cortical NuMA enrichment. (A) Sequence alignments of NuMA amino acid sequence near T2055 from various NuMA orthologs. A red asterisk marks the Cdk1 phosphorylation site, and blue asterisks show basic amino acids. (B) Schematic representation of wild-type fragment of NuMA(1411–2115) tagged with GFP at the N-terminus, and several mutant forms where either two or four basic residues were mutated to alanine (2KK>A, 4KR>A), or where the conserved threonine residue was mutated to serine (T>S). Asterisks indicate T2055; MTs, region mediating interaction with microtubules; NLS, nuclear localization signal. (C–F) Images from time-lapse microscopy of HeLa cells stably expressing mCherry–H2B and were transfected with GFP–NuMA(1411–2115) (C), GFP–NuMA(1411–2115)2KK>A (D), GFP–NuMA(1411–2115)4KR>A (E), and GFP–NuMA(1411–2115)T>S (F). The GFP signal is shown in green and mCherry signal is in magenta. Note the significant loss of cortical NuMA in cells expressing GFP–NuMA(1411–2115)2KK>A or GFP–NuMA(1411–2115)4KR>A. Also, note the dramatically weaker cortical GFP signal in cells expressing GFP–NuMA(1411–2115)T>S. Arrows indicate cortical GFP localization and boxes indicate examples of regions used for quantification. (G) Quantification of cortical enrichment for cells that underwent metaphase to anaphase transition, as shown in C–E (see Materials and Methods for detail). Data show mean±s.d. for n>5 cells, with individual data points plotted. GFP–NuMA(1411–2115) versus GFP–NuMA(1411–2115)2KK>A; P=0.28, 0.83, 0.032, 0.006, 0.023 and 0.068 for 0, 3, 6, 9, 12 and 15 min, respectively. GFP– NuMA(1411–2115) versus GFP–NuMA(1411–2115)4KR>A; P=0.0003 at 0 min, P<0.0001 at 3–15 min timepoints (two-tailed Student's t-test). (H) Quantification of cortical enrichment for cells that underwent metaphase to anaphase transition, as shown in C and F. Data show mean±s.d. for ≥18 cells, with individual data points plotted. GFP–NuMA(1411–2115) versus GFP–NuMA(1411–2115)T>S; P=0.89, 0.04, 0.002, <0.0001, 0.18 and 0.35 for 0, 3, 6, 9, 12 and15 min, respectively. (I–L) Images from time-lapse microscopy of HeLa Kyoto cells stably expressing mCherry–H2B (I), stably expressing mCherry–H2B and depleted of endogenous NuMA (J), stably co-expressing AcGFP–NuMA and mCherry–H2B and depleted of endogenous NuMA (K), or stably co-expressing AcGFP–NuMA4KR>A and mCherry–H2B and depleted of endogenous NuMA (L). The GFP signal is shown in green, the mCherry signal is in magenta. (M) Schematic representation for the calculation of the distance [d] between inter-chromatids in cells that underwent anaphase progression. (N) Quantification of inter-chromatid distance in cells as shown in I–L. Data are mean±s.d. inter-chromatid distances during anaphase progression for more than 15 cells in each condition (see Materials and Methods). **P=0.0002 at 2 min, ***P<0.0001 for 4–10 min timepoints between AcGFP–NuMA- and AcGFP–NuMA4K>R-expressing cells upon depletion of endogenous NuMA (two-tailed Student's t-test).

Article Snippet: For immunoprecipitation, 3 mg of cell lysates procured from the nocodazole (100 nM)-arrested mitotic HeLa Kyoto cells expressing either AcGFP-tagged or GFP-tagged proteins were incubated with 30 μl GFP-Trap agarose beads (Chromotek, ACT-CM-GFA0050) in lysis buffer [50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 25 mM sodium fluoride, 0.1 mM sodium orthovanadate (Sigma-Aldrich, S6508), 0.1 mM PMSF (Calbiochem, 7110), 0.2% Triton-X100, 0.3% NP-40, 100 nM okadaic acid and Complete EDTA-free protease inhibitor (Merck, 539134)] for 4 h at 4°C.

Techniques: Sequencing, Mutagenesis, Time-lapse Microscopy, Stable Transfection, Expressing, Transfection, Two Tailed Test

Journal: Scientific Reports

Article Title: Evidence for association of Vibrio echinoideorum with tissue necrosis on test of the green sea urchin Strongylocentrotus droebachiensis

doi: 10.1038/s41598-022-08772-2

Figure Lengend Snippet: Overview of putative virulence genes identified in the V. echinoideorum genome.

Article Snippet: traA, traB, traC, traD, traE, traF, traH, traI, traK, traL, traN, traU, traV traW, , IncF plasmid conjugative transfer protein.

Techniques: Chemotaxis Assay, Binding Assay, Membrane, Plasmid Preparation, Transduction

Journal: Pest Management Science

Article Title: Testing an integrated approach for managing roof rats in citrus orchards

doi: 10.1002/ps.8672

Figure Lengend Snippet: The mean percent efficacy (percent change in roof rat visitation across respective monitoring periods with positive efficacy representative of a reduction in rat activity) and associated standard errors of bait station, integrated pest management (IPM) and untreated plots (Control) for roof rat control within four citrus orchards in the southern San Joaquin Valley, California, from spring 2022 to spring 2023 (Trial 1). We applied 0.005% diphacinone‐treated oats in elevated bait stations for bait station and IPM plots, with efficacy after this baiting period represented as ‘Post‐bait’. For IPM plots, we followed up bait application with snap traps and Goodnature® A24 traps. We conducted additional indices of roof rat activity 2‐months and 5‐months post‐bait application to detect differences over the duration of the 6‐month trial. Significant differences across treatment types within a specified time period are denoted by different letters.

Article Snippet: Additionally, the recent introduction of the Goodnature® A24 trap (Goodnature Ltd, Wellington, NZ; hereafter A24 traps) into the U.S. has the potential to increase the utility of trapping as a management tool, as each A24 trap allows for the capture of up to 24 rats without the need to check or reset over a 4–6‐month period.

Techniques: Activity Assay, Control