rabbit polyclonal anti pds5a antibodies  (Proteintech)

Journal: Science advances

Article Title: The cohesin-associated protein Pds5A governs the meiotic spindle assembly via deubiquitination of Kif5B in oocytes.

doi: 10.1126/sciadv.adt6159

Figure Lengend Snippet: Fig. 1. Subcellular localization and protein expression patterns of Pds5A during mouse oocyte meiotic maturation. (A) Fluorescence images of Pds5A localization in oocytes. Mouse oocytes at different developmental stages were costained with Pds5A (Santa Cruz Biotechnology) and α-tubulin antibodies and counterstained with Hoechst 33342. Scale bar, 20 μm. (B) Fluorescence intensity profiles of Pds5A and α-tubulin along the yellow line in (A). (C) Immunoblotting analysis of Pds5A protein levels during oocyte meiosis corresponding to GV, GVBD, MI, and MII stages. The blots were probed with Pds5A (Proteintech) and β-actin antibodies, respectively. (D) Quantification of Pds5A protein levels during oocyte meiosis. The band intensity of Pds5A was normalized with that of β-actin. A.U., arbitrary unit.

Article Snippet: Rabbit polyclonal anti- Pds5A antibodies were purchased from Proteintech (Rosemont, IL, USA; catalog no.: 17485- 1- AP) and Bethyl Laboratories (Montgomery, TX, USA; catalog no.: A300- 089A- T); mouse monoclonal anti- Pds5A antibody was purchased from Santa Cruz Biotechnology (Dallas, Texas, USA; catalog no.: sc- 515263); mouse monoclonal anti–α- tubulin–fluorescein isothiocyanate (FITC) antibody was purchased from Sigma- Aldrich (St. Louis, MO, USA; catalog no.: F2168); sheep polyclonal anti- BubR1 antibody was purchased from Abcam (Cambridge, MA, USA; catalog no.: ab28193); rabbit polyclonal anti- Mad2L1 antibody was purchased from Proteintech (catalog no.: 10337- 1- AP); human anti- centromere antibody was purchased from Antibodies Incorporated (Davis, CA, USA; catalog no.: CA95617); rabbit polyclonal anti- Kif5B antibody was purchased from ABclonal Technology (Wuhan, China; catalog no.: A15284); mouse monoclonal anti- Kif5B antibody was purchased from Santa Cruz Biotechnology (catalog no.: sc- 133184); mouse monoclonal anti–β- actin antibody was purchased from Proteintech (catalog no.: 60008- 1- Ig); mouse monoclonal anti- Usp14 antibody was purchased from Santa Cruz Biotechnology (catalog no.: sc- 398009); rabbit polyclonal anti- Usp14 antibody was purchased from Proteintech (catalog no.: 14517- 1- AP); rabbit monoclonal anti- Rec8 antibody was purchased from Abcam (catalog no.: ab192241); mouse monoclonal anti- Rad21 antibody was purchased from Sigma- Aldrich (catalog no.: 05- 908); rabbit polyclonal anti- HA antibody was purchased from Proteintech (catalog no.: 51064- 2- AP); rabbit monoclonal anti- Myc antibody was purchased from ABclonal Technology (catalog no.: AE070).

Techniques: Expressing, Fluorescence, Western Blot

Journal: Science advances

Article Title: The cohesin-associated protein Pds5A governs the meiotic spindle assembly via deubiquitination of Kif5B in oocytes.

doi: 10.1126/sciadv.adt6159

Figure Lengend Snippet: Fig. 2. Effects of Pds5A depletion on the mouse oocyte meiotic progression. (A) Representative images of GVBD oocytes observed at 4 hours following release from 3-isobutyl-1-metyl-xanthine in control and Pds5A-MO groups. Scale bar, 80 μm. (B) The rate of GVBD in control (n = 169) and Pds5A-MO (n = 185) oocytes. (C) Representa- tive images of MII oocytes observed at 10 hours post-GVBD in control, Pds5A-MO, and Pds5A-rescue (Pds5A-MO + Pds5A-cRNA) groups. Scale bar, 80 μm. (D) The rate of PBE in control (n = 119), Pds5A-MO (n = 119), and Pds5A-rescue (n = 113) oocytes. (E) Representative images of chromosome morphology in Pds5A-MO oocytes without PB1 after maturation. Scale bar, 5 μm. (F) The percentage of oocytes arresting at different developmental stages in Pds5A-MO (n = 76) group. (G) Fluorescence images of BubR1 present on the chromosomes in control and Pds5A-MO oocytes at Pro-MI and MI stages. Scale bar, 5 μm. (H) The proportion of BubR1 presence on the chromo- somes in control (n = 73) and Pds5A-MO (n = 69) oocytes at MI stage. (I) Fluorescence images of Mad2 present on the chromosomes in control and Pds5A-MO oocytes at Pro-MI and MI stages. Scale bar, 5 μm. (J) The proportion of Mad2 presence on the chromosomes in control (n = 71) and Pds5A-MO (n = 86) oocytes at MI stage. Data in (B), (D), (F), (H), and (J) were designated as mean percentage (means ± SEM) of at least three independent experiments. **P < 0.01; ns, no significance.

Article Snippet: Rabbit polyclonal anti- Pds5A antibodies were purchased from Proteintech (Rosemont, IL, USA; catalog no.: 17485- 1- AP) and Bethyl Laboratories (Montgomery, TX, USA; catalog no.: A300- 089A- T); mouse monoclonal anti- Pds5A antibody was purchased from Santa Cruz Biotechnology (Dallas, Texas, USA; catalog no.: sc- 515263); mouse monoclonal anti–α- tubulin–fluorescein isothiocyanate (FITC) antibody was purchased from Sigma- Aldrich (St. Louis, MO, USA; catalog no.: F2168); sheep polyclonal anti- BubR1 antibody was purchased from Abcam (Cambridge, MA, USA; catalog no.: ab28193); rabbit polyclonal anti- Mad2L1 antibody was purchased from Proteintech (catalog no.: 10337- 1- AP); human anti- centromere antibody was purchased from Antibodies Incorporated (Davis, CA, USA; catalog no.: CA95617); rabbit polyclonal anti- Kif5B antibody was purchased from ABclonal Technology (Wuhan, China; catalog no.: A15284); mouse monoclonal anti- Kif5B antibody was purchased from Santa Cruz Biotechnology (catalog no.: sc- 133184); mouse monoclonal anti–β- actin antibody was purchased from Proteintech (catalog no.: 60008- 1- Ig); mouse monoclonal anti- Usp14 antibody was purchased from Santa Cruz Biotechnology (catalog no.: sc- 398009); rabbit polyclonal anti- Usp14 antibody was purchased from Proteintech (catalog no.: 14517- 1- AP); rabbit monoclonal anti- Rec8 antibody was purchased from Abcam (catalog no.: ab192241); mouse monoclonal anti- Rad21 antibody was purchased from Sigma- Aldrich (catalog no.: 05- 908); rabbit polyclonal anti- HA antibody was purchased from Proteintech (catalog no.: 51064- 2- AP); rabbit monoclonal anti- Myc antibody was purchased from ABclonal Technology (catalog no.: AE070).

Techniques: Control, Fluorescence

Journal: Science advances

Article Title: The cohesin-associated protein Pds5A governs the meiotic spindle assembly via deubiquitination of Kif5B in oocytes.

doi: 10.1126/sciadv.adt6159

Figure Lengend Snippet: Fig. 3. Effects of Pds5A depletion on the spindle organization and chromosome alignment in mouse oocytes. (A) Fluorescence images of spindle morphology and chromosome alignment in control, Pds5A-MO, and Pds5A-rescue oocytes at MI stage. The yellow arrow points to the misaligned chromosomes. Scale bar, 20 μm. (B) The rate of abnormal spindles in control (n = 72), Pds5A-MO (n = 73), and Pds5A-rescue (n = 80) oocytes at MI stage. (C) Representative images showing different types of spindle defects observed in Pds5A-MO oocytes at MI stage. Scale bar, 10 μm. (D) The number of different types of spindle defects in Pds5A-MO (n = 73) oocytes at MI stage. (E) Schematic picture showing how the spindle length, width, and pole width were determined. (F) The ratio of spindle length to width in control (n = 23), Pds5A-MO (n = 21), and Pds5A-rescue (n = 22) oocytes at MI stage. (G) The rate of misaligned bivalents in control (n = 72), Pds5A-MO (n = 73), and Pds5A-rescue (n = 80) oocytes at MI stage. (H) Quantification of MI plate width in control (n = 24), Pds5A-MO (n = 24), and Pds5A-rescue (n = 22) oocytes at MI stage. Data in (B) and (G) were designated as mean percentage (means ± SEM), and (F) and (H) were designated as mean value (means ± SD) of at least three independent experiments. **P < 0.01; ***P < 0.001.

Article Snippet: Rabbit polyclonal anti- Pds5A antibodies were purchased from Proteintech (Rosemont, IL, USA; catalog no.: 17485- 1- AP) and Bethyl Laboratories (Montgomery, TX, USA; catalog no.: A300- 089A- T); mouse monoclonal anti- Pds5A antibody was purchased from Santa Cruz Biotechnology (Dallas, Texas, USA; catalog no.: sc- 515263); mouse monoclonal anti–α- tubulin–fluorescein isothiocyanate (FITC) antibody was purchased from Sigma- Aldrich (St. Louis, MO, USA; catalog no.: F2168); sheep polyclonal anti- BubR1 antibody was purchased from Abcam (Cambridge, MA, USA; catalog no.: ab28193); rabbit polyclonal anti- Mad2L1 antibody was purchased from Proteintech (catalog no.: 10337- 1- AP); human anti- centromere antibody was purchased from Antibodies Incorporated (Davis, CA, USA; catalog no.: CA95617); rabbit polyclonal anti- Kif5B antibody was purchased from ABclonal Technology (Wuhan, China; catalog no.: A15284); mouse monoclonal anti- Kif5B antibody was purchased from Santa Cruz Biotechnology (catalog no.: sc- 133184); mouse monoclonal anti–β- actin antibody was purchased from Proteintech (catalog no.: 60008- 1- Ig); mouse monoclonal anti- Usp14 antibody was purchased from Santa Cruz Biotechnology (catalog no.: sc- 398009); rabbit polyclonal anti- Usp14 antibody was purchased from Proteintech (catalog no.: 14517- 1- AP); rabbit monoclonal anti- Rec8 antibody was purchased from Abcam (catalog no.: ab192241); mouse monoclonal anti- Rad21 antibody was purchased from Sigma- Aldrich (catalog no.: 05- 908); rabbit polyclonal anti- HA antibody was purchased from Proteintech (catalog no.: 51064- 2- AP); rabbit monoclonal anti- Myc antibody was purchased from ABclonal Technology (catalog no.: AE070).

Techniques: Fluorescence, Control

Journal: Science advances

Article Title: The cohesin-associated protein Pds5A governs the meiotic spindle assembly via deubiquitination of Kif5B in oocytes.

doi: 10.1126/sciadv.adt6159

Figure Lengend Snippet: Fig. 4. Effects of Pds5A depletion on the K-M attachment and chromosome ploidy in mouse oocytes. (A) Fluorescence images kinetochores and microtubule fibers in control, Pds5A-MO, and Pds5A-rescue oocytes at MI stage. White arrows indicate unattached kinetochores. Scale bars, 5 and 2.5 μm. (B) The percentage of unattached kinetochores in control (n = 90), Pds5A-MO, (n = 125), and Pds5A- rescue (n = 103) oocytes at MI stage. (C) Representative images of chromosome spreads in control, Pds5A-MO, and Pds5A-rescue oocytes at MII stage. Scale bar, 8 μm. (D) The percentage of aneuploid oocytes at MII stage in control (n = 76), Pds5A-MO (n = 78), and Pds5A-rescue (n = 76) groups. Data in (B) and (D) were designated as mean percentage (means ± SEM) of at least three independent ex- periments. **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: Rabbit polyclonal anti- Pds5A antibodies were purchased from Proteintech (Rosemont, IL, USA; catalog no.: 17485- 1- AP) and Bethyl Laboratories (Montgomery, TX, USA; catalog no.: A300- 089A- T); mouse monoclonal anti- Pds5A antibody was purchased from Santa Cruz Biotechnology (Dallas, Texas, USA; catalog no.: sc- 515263); mouse monoclonal anti–α- tubulin–fluorescein isothiocyanate (FITC) antibody was purchased from Sigma- Aldrich (St. Louis, MO, USA; catalog no.: F2168); sheep polyclonal anti- BubR1 antibody was purchased from Abcam (Cambridge, MA, USA; catalog no.: ab28193); rabbit polyclonal anti- Mad2L1 antibody was purchased from Proteintech (catalog no.: 10337- 1- AP); human anti- centromere antibody was purchased from Antibodies Incorporated (Davis, CA, USA; catalog no.: CA95617); rabbit polyclonal anti- Kif5B antibody was purchased from ABclonal Technology (Wuhan, China; catalog no.: A15284); mouse monoclonal anti- Kif5B antibody was purchased from Santa Cruz Biotechnology (catalog no.: sc- 133184); mouse monoclonal anti–β- actin antibody was purchased from Proteintech (catalog no.: 60008- 1- Ig); mouse monoclonal anti- Usp14 antibody was purchased from Santa Cruz Biotechnology (catalog no.: sc- 398009); rabbit polyclonal anti- Usp14 antibody was purchased from Proteintech (catalog no.: 14517- 1- AP); rabbit monoclonal anti- Rec8 antibody was purchased from Abcam (catalog no.: ab192241); mouse monoclonal anti- Rad21 antibody was purchased from Sigma- Aldrich (catalog no.: 05- 908); rabbit polyclonal anti- HA antibody was purchased from Proteintech (catalog no.: 51064- 2- AP); rabbit monoclonal anti- Myc antibody was purchased from ABclonal Technology (catalog no.: AE070).

Techniques: Fluorescence, Control

Journal: Science advances

Article Title: The cohesin-associated protein Pds5A governs the meiotic spindle assembly via deubiquitination of Kif5B in oocytes.

doi: 10.1126/sciadv.adt6159

Figure Lengend Snippet: Fig. 5. The oocyte maturation and female fertility in Pds5A+/− mice. (A) Representative images of pups from WT and Pds5A+/− female mice. Scale bar, 1 cm. (B) The average number of pups per litter from WT (n = 9) and Pds5A+/− (n = 8) female mice. (C) Representative images of superovulated oocytes from WT and Pds5A+/− female mice. Scale bar, 80 μm. (D to F) The number, PBE rate, and death rate of superovulated oocytes in WT (n = 248) and Pds5A+/− (n = 173) groups. (G) Fluorescence images of spindles and chromosomes in WT and Pds5A+/− superovulated oocytes. Scale bars, 20 and 10 μm. (H) The rate of abnormal spindles in WT (n = 85) and Pds5A+/− (n = 84) superovulated oocytes. (I) The number of different defective spindles in Pds5A+/− (n = 40) superovulated oocytes. (J) The rate of misaligned chromosomes in WT (n = 85) and Pds5A+/− (n = 84) superovulated oocytes. (K) Representative images of chromosome spreads in WT and Pds5A+/− superovulated oocytes. Scale bar, 10 μm. (L) The percentage of aneuploid oocytes in WT (n = 84) and Pds5A+/− (n = 57) groups. (M) Fluorescence images of spindles and chromosomes in WT and Pds5A+/− MI oocytes. Scale bar, 20 μm. (N) The rate of abnormal spindles in WT (n = 100) and Pds5A+/− (n = 118) MI oocytes. (O) The number of different defective spindles in Pds5A+/− (n = 36) MI oocytes. (P) The rate of misaligned bivalents in WT (n = 100) and Pds5A+/− (n = 118) MI oocytes. Data in (B) and (D) were designated as means ± SD, and (E), (F), (H), (J), (L), (N), and (P) were designated as means ± SEM of at least three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: Rabbit polyclonal anti- Pds5A antibodies were purchased from Proteintech (Rosemont, IL, USA; catalog no.: 17485- 1- AP) and Bethyl Laboratories (Montgomery, TX, USA; catalog no.: A300- 089A- T); mouse monoclonal anti- Pds5A antibody was purchased from Santa Cruz Biotechnology (Dallas, Texas, USA; catalog no.: sc- 515263); mouse monoclonal anti–α- tubulin–fluorescein isothiocyanate (FITC) antibody was purchased from Sigma- Aldrich (St. Louis, MO, USA; catalog no.: F2168); sheep polyclonal anti- BubR1 antibody was purchased from Abcam (Cambridge, MA, USA; catalog no.: ab28193); rabbit polyclonal anti- Mad2L1 antibody was purchased from Proteintech (catalog no.: 10337- 1- AP); human anti- centromere antibody was purchased from Antibodies Incorporated (Davis, CA, USA; catalog no.: CA95617); rabbit polyclonal anti- Kif5B antibody was purchased from ABclonal Technology (Wuhan, China; catalog no.: A15284); mouse monoclonal anti- Kif5B antibody was purchased from Santa Cruz Biotechnology (catalog no.: sc- 133184); mouse monoclonal anti–β- actin antibody was purchased from Proteintech (catalog no.: 60008- 1- Ig); mouse monoclonal anti- Usp14 antibody was purchased from Santa Cruz Biotechnology (catalog no.: sc- 398009); rabbit polyclonal anti- Usp14 antibody was purchased from Proteintech (catalog no.: 14517- 1- AP); rabbit monoclonal anti- Rec8 antibody was purchased from Abcam (catalog no.: ab192241); mouse monoclonal anti- Rad21 antibody was purchased from Sigma- Aldrich (catalog no.: 05- 908); rabbit polyclonal anti- HA antibody was purchased from Proteintech (catalog no.: 51064- 2- AP); rabbit monoclonal anti- Myc antibody was purchased from ABclonal Technology (catalog no.: AE070).

Techniques: Fluorescence

Journal: Science advances

Article Title: The cohesin-associated protein Pds5A governs the meiotic spindle assembly via deubiquitination of Kif5B in oocytes.

doi: 10.1126/sciadv.adt6159

Figure Lengend Snippet: Fig. 6. Identification of binding partners of Pds5A. (A) Representative bind- ing candidates of Pds5A as shown in MS data. (B) Costaining of Pds5A and Kif5B in oocytes at MI stage. Scale bars, 20 and 5 μm. (C) Fluorescence intensity pro- files of Pds5A and Kif5B along the white line. (D) Co-IP of Pds5A and Kif5B as precipitated with Pds5A antibody. The blots of precipitates were probed with Kif5B and Pds5A antibodies, respectively. (E) Co-IP of Pds5A and Kif5B as pre- cipitated with Kif5B antibody. The blots of precipitates were probed with Pds5A and Kif5B antibodies, respectively.

Article Snippet: Rabbit polyclonal anti- Pds5A antibodies were purchased from Proteintech (Rosemont, IL, USA; catalog no.: 17485- 1- AP) and Bethyl Laboratories (Montgomery, TX, USA; catalog no.: A300- 089A- T); mouse monoclonal anti- Pds5A antibody was purchased from Santa Cruz Biotechnology (Dallas, Texas, USA; catalog no.: sc- 515263); mouse monoclonal anti–α- tubulin–fluorescein isothiocyanate (FITC) antibody was purchased from Sigma- Aldrich (St. Louis, MO, USA; catalog no.: F2168); sheep polyclonal anti- BubR1 antibody was purchased from Abcam (Cambridge, MA, USA; catalog no.: ab28193); rabbit polyclonal anti- Mad2L1 antibody was purchased from Proteintech (catalog no.: 10337- 1- AP); human anti- centromere antibody was purchased from Antibodies Incorporated (Davis, CA, USA; catalog no.: CA95617); rabbit polyclonal anti- Kif5B antibody was purchased from ABclonal Technology (Wuhan, China; catalog no.: A15284); mouse monoclonal anti- Kif5B antibody was purchased from Santa Cruz Biotechnology (catalog no.: sc- 133184); mouse monoclonal anti–β- actin antibody was purchased from Proteintech (catalog no.: 60008- 1- Ig); mouse monoclonal anti- Usp14 antibody was purchased from Santa Cruz Biotechnology (catalog no.: sc- 398009); rabbit polyclonal anti- Usp14 antibody was purchased from Proteintech (catalog no.: 14517- 1- AP); rabbit monoclonal anti- Rec8 antibody was purchased from Abcam (catalog no.: ab192241); mouse monoclonal anti- Rad21 antibody was purchased from Sigma- Aldrich (catalog no.: 05- 908); rabbit polyclonal anti- HA antibody was purchased from Proteintech (catalog no.: 51064- 2- AP); rabbit monoclonal anti- Myc antibody was purchased from ABclonal Technology (catalog no.: AE070).

Techniques: Binding Assay, Fluorescence, Co-Immunoprecipitation Assay

Journal: Science advances

Article Title: The cohesin-associated protein Pds5A governs the meiotic spindle assembly via deubiquitination of Kif5B in oocytes.

doi: 10.1126/sciadv.adt6159

Figure Lengend Snippet: Fig. 7. Recruitment of Usp14 by Pds5A stabilizes Kif5B in mouse oocytes. (A) Immunoblotting analysis showing the protein levels of Kif5B in control, Pds5A-MO, and Pds5A-rescue oocytes. (B) Fluorescence images of Kif5B in control, Pds5A-MO, and Pds5A-rescue oocytes at MI stage. Scale bars, 20 and 5 μm. (C) The fluorescence inten- sity of Kif5B signals in control (n = 19), Pds5A-MO (n = 20), and Pds5A-rescue (n = 19) oocytes at MI stage. (D) qRT-PCR analysis showing the mRNA levels of Kif5B in control (n = 30) and Pds5A-MO (n = 30) oocytes. (E) Immunoblotting analysis showing the protein levels of Kif5B in control, Pds5A-MO, and Pds5A-MO+MG132 (10 μM) oocytes. (F) Representative binding DUBs of Pds5A as shown in MS data. (G to K) Immunoblotting analysis showing the protein levels of Kif5B in control, PR-619-treated, WP1130- treated, IU1-treated, USP5-IN-1-treated, and FT671-treated oocytes, respectively. PR-619 (10 μM), 10 μM WP1130, 25 μM IU1, 10 μM USP5-IN-1, and 10 μM FT671 were used to treat GV oocytes for 22 hours, respectively. (L) Co-IP of Pds5A and Usp14 as precipitated with Pds5A antibody. (M) Costaining of Pds5A and Usp14 in oocytes at MI stage. Scale bars, 20 and 5 μm. (N) Fluorescence intensity profiles of Pds5A and Usp14 along the white line in (M). (O) Immunoblotting analysis showing the protein levels of Usp14 in control and Pds5A-MO oocytes. (P) Fluorescence images of Usp14 in control and Pds5A-MO oocytes at MI stage. Scale bar, 5 μm. (Q) The fluorescence intensity of Usp14 signals in control (n = 26) and Pds5A-MO (n = 35) oocytes at MI stage. Data in (D) were designated as mean percentage (means ± SEM), and (C) and (Q) were designated as mean value (means ± SD) of at least three independent experiments. ***P < 0.001; ****P < 0.0001; ns, no significance.

Article Snippet: Rabbit polyclonal anti- Pds5A antibodies were purchased from Proteintech (Rosemont, IL, USA; catalog no.: 17485- 1- AP) and Bethyl Laboratories (Montgomery, TX, USA; catalog no.: A300- 089A- T); mouse monoclonal anti- Pds5A antibody was purchased from Santa Cruz Biotechnology (Dallas, Texas, USA; catalog no.: sc- 515263); mouse monoclonal anti–α- tubulin–fluorescein isothiocyanate (FITC) antibody was purchased from Sigma- Aldrich (St. Louis, MO, USA; catalog no.: F2168); sheep polyclonal anti- BubR1 antibody was purchased from Abcam (Cambridge, MA, USA; catalog no.: ab28193); rabbit polyclonal anti- Mad2L1 antibody was purchased from Proteintech (catalog no.: 10337- 1- AP); human anti- centromere antibody was purchased from Antibodies Incorporated (Davis, CA, USA; catalog no.: CA95617); rabbit polyclonal anti- Kif5B antibody was purchased from ABclonal Technology (Wuhan, China; catalog no.: A15284); mouse monoclonal anti- Kif5B antibody was purchased from Santa Cruz Biotechnology (catalog no.: sc- 133184); mouse monoclonal anti–β- actin antibody was purchased from Proteintech (catalog no.: 60008- 1- Ig); mouse monoclonal anti- Usp14 antibody was purchased from Santa Cruz Biotechnology (catalog no.: sc- 398009); rabbit polyclonal anti- Usp14 antibody was purchased from Proteintech (catalog no.: 14517- 1- AP); rabbit monoclonal anti- Rec8 antibody was purchased from Abcam (catalog no.: ab192241); mouse monoclonal anti- Rad21 antibody was purchased from Sigma- Aldrich (catalog no.: 05- 908); rabbit polyclonal anti- HA antibody was purchased from Proteintech (catalog no.: 51064- 2- AP); rabbit monoclonal anti- Myc antibody was purchased from ABclonal Technology (catalog no.: AE070).

Techniques: Western Blot, Control, Fluorescence, Quantitative RT-PCR, Binding Assay, Co-Immunoprecipitation Assay

Journal: Cell reports methods

Article Title: Microscopy-based single-cell proteomic profiling reveals heterogeneity in DNA damage response dynamics.

doi: 10.1016/j.crmeth.2022.100237

Figure Lengend Snippet: Figure 4. Single-cell quantitative proteomics on group 1 and group 2 cells collected by the FUNpro pipeline (A) Phototagging of the group 2 cells. Scale bar: 500 mm. The zoomed-in images showed a representative cell (arrow) before and after phototagging. Scale bar: 50 mm. Red, 53BP1; green, phototagging (PT) dye. (B) Schematic protocol of single-cell proteomics analysis. Four non-tagged cells, four tagged cells, and 200 non-tagged cells (serving as carrier cells) were labeled with respective 10-plex TMT labels as indicated and then mixed into one sample before being subjected to liquid chromatography tandem mass spectrometry (LC-MS/MS). Ten samples, in total 40 group 1 and 40 group 2 cells, were analyzed. (C) (i) A volcano plot showing proteins enriched either in group 1 (normal DDR) or group 2 (abnormal DDR) cells. Together, 80 cells were pooled into the analysis. Dashed lines show the cutoff value of FDR at 0.05. (ii) DDR-related protein, PDS5A, was found upregulated in group 2 cells.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit polyclonal anti-PDS5A Novus Cat#NBP1-87904; RRID: AB_11015600 Rabbit polyclonal anti-gH2AX Abcam Cat#ab11174; RRID: AB_297813 Goat polyclonal Alexa-488 anti-rabbit Abcam Cat#ab150077; RRID: AB_2630356 Chemicals, peptides, and recombinant proteins DMEM medium Thermo Fisher Cat#11965092 Lipofectamine 3000 Transfection Reagent Thermo Fisher Cat#L3000001 Fetal bovine serum Thermo Fisher Cat#A4736301 Penicillin-Streptomycin Thermo Fisher Cat#15140122 FluoroBrite DMEM medium Thermo Fisher Cat#A1896701 PA Janelia Fluor 646, SE Tocris Bioscience Cat#6150 Triethylammonium bicarbonate buffer Sigma-Aldrich Cat#T7408 Water, LC-MS Grade Thermo Fisher Cat#51140 Acetonitrile (ACN), LC-MS Grade Thermo Fisher Cat#51101 TMT10plex Isobaric Label Reagent Set Thermo Fisher Cat#90110 Hydroxylamine solution Sigma-Aldrich Cat#467804 Formic acid Sigma-Aldrich Cat#33015-M Deposited data Raw and analyzed data This paper ProteomeXchange: PXD034370 Experimental models: Cell lines Human: U2OS cells transfected with PBmScarlet-53BP1 Erasmus University Medical Center, Molecular Genetics N/A Software and algorithms Automatic cell tracking analysis You et al. (2022) https://sourceforge.net/projects/funseq/ ImageJ Schneider et al. (2012) https://imagej.nih.gov/ij/ Matlab MathWorks https://nl.mathworks.com/ products/matlab.html MaxQuant Cox and Mann (2008) https://www.maxquant.org/ Perseus Tyanova et al. (2016) https://www.maxquant.org/perseus/ Other Orbitrap Eclipse Tribid mass spectrometer Thermo Fisher http://thermofisher.com/OrbitrapEclipse

Techniques: Quantitative Proteomics, Labeling, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy

Journal: Cell reports methods

Article Title: Microscopy-based single-cell proteomic profiling reveals heterogeneity in DNA damage response dynamics.

doi: 10.1016/j.crmeth.2022.100237

Figure Lengend Snippet: Figure 5. Immunofluorescence validation of the PDS5A protein on group 1 and group 2 (with asterisk) cells (A) (i) A UFO image of U2OS cells expressing 53BP1-mScarlet (gray). The image was taken after 24 h of IR. Scale bar: 10 mm. (ii) A confocal image of immu- nofluorescence against the PDS5A protein (16 colored) from the same fieldof-view. Scale bar: 10 mm. (B) Immunofluorescence quantification of the PDS5A protein on group 1 and group 2 cells. In total, 16 pairs of cells were analyzed with p value lower than 0.01 (Student’s t test).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit polyclonal anti-PDS5A Novus Cat#NBP1-87904; RRID: AB_11015600 Rabbit polyclonal anti-gH2AX Abcam Cat#ab11174; RRID: AB_297813 Goat polyclonal Alexa-488 anti-rabbit Abcam Cat#ab150077; RRID: AB_2630356 Chemicals, peptides, and recombinant proteins DMEM medium Thermo Fisher Cat#11965092 Lipofectamine 3000 Transfection Reagent Thermo Fisher Cat#L3000001 Fetal bovine serum Thermo Fisher Cat#A4736301 Penicillin-Streptomycin Thermo Fisher Cat#15140122 FluoroBrite DMEM medium Thermo Fisher Cat#A1896701 PA Janelia Fluor 646, SE Tocris Bioscience Cat#6150 Triethylammonium bicarbonate buffer Sigma-Aldrich Cat#T7408 Water, LC-MS Grade Thermo Fisher Cat#51140 Acetonitrile (ACN), LC-MS Grade Thermo Fisher Cat#51101 TMT10plex Isobaric Label Reagent Set Thermo Fisher Cat#90110 Hydroxylamine solution Sigma-Aldrich Cat#467804 Formic acid Sigma-Aldrich Cat#33015-M Deposited data Raw and analyzed data This paper ProteomeXchange: PXD034370 Experimental models: Cell lines Human: U2OS cells transfected with PBmScarlet-53BP1 Erasmus University Medical Center, Molecular Genetics N/A Software and algorithms Automatic cell tracking analysis You et al. (2022) https://sourceforge.net/projects/funseq/ ImageJ Schneider et al. (2012) https://imagej.nih.gov/ij/ Matlab MathWorks https://nl.mathworks.com/ products/matlab.html MaxQuant Cox and Mann (2008) https://www.maxquant.org/ Perseus Tyanova et al. (2016) https://www.maxquant.org/perseus/ Other Orbitrap Eclipse Tribid mass spectrometer Thermo Fisher http://thermofisher.com/OrbitrapEclipse

Techniques: Biomarker Discovery, Expressing

Journal: Cell reports methods

Article Title: Microscopy-based single-cell proteomic profiling reveals heterogeneity in DNA damage response dynamics.

doi: 10.1016/j.crmeth.2022.100237

Figure Lengend Snippet: Figure 4. Single-cell quantitative proteomics on group 1 and group 2 cells collected by the FUNpro pipeline (A) Phototagging of the group 2 cells. Scale bar: 500 mm. The zoomed-in images showed a representative cell (arrow) before and after phototagging. Scale bar: 50 mm. Red, 53BP1; green, phototagging (PT) dye. (B) Schematic protocol of single-cell proteomics analysis. Four non-tagged cells, four tagged cells, and 200 non-tagged cells (serving as carrier cells) were labeled with respective 10-plex TMT labels as indicated and then mixed into one sample before being subjected to liquid chromatography tandem mass spectrometry (LC-MS/MS). Ten samples, in total 40 group 1 and 40 group 2 cells, were analyzed. (C) (i) A volcano plot showing proteins enriched either in group 1 (normal DDR) or group 2 (abnormal DDR) cells. Together, 80 cells were pooled into the analysis. Dashed lines show the cutoff value of FDR at 0.05. (ii) DDR-related protein, PDS5A, was found upregulated in group 2 cells.

Article Snippet: The cells were then incubated with 1:500 rabbit polyclonal anti-PDS5A antibody (Novus) or 1:1000 rabbit polyclonal anti-gH2AX antibody (Abcam) for 90 min at room temperature.

Techniques: Quantitative Proteomics, Labeling, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy

Journal: Cell reports methods

Article Title: Microscopy-based single-cell proteomic profiling reveals heterogeneity in DNA damage response dynamics.

doi: 10.1016/j.crmeth.2022.100237

Figure Lengend Snippet: Figure 5. Immunofluorescence validation of the PDS5A protein on group 1 and group 2 (with asterisk) cells (A) (i) A UFO image of U2OS cells expressing 53BP1-mScarlet (gray). The image was taken after 24 h of IR. Scale bar: 10 mm. (ii) A confocal image of immu- nofluorescence against the PDS5A protein (16 colored) from the same fieldof-view. Scale bar: 10 mm. (B) Immunofluorescence quantification of the PDS5A protein on group 1 and group 2 cells. In total, 16 pairs of cells were analyzed with p value lower than 0.01 (Student’s t test).

Article Snippet: The cells were then incubated with 1:500 rabbit polyclonal anti-PDS5A antibody (Novus) or 1:1000 rabbit polyclonal anti-gH2AX antibody (Abcam) for 90 min at room temperature.

Techniques: Biomarker Discovery, Expressing

Journal: Cell Reports Methods

Article Title: Microscopy-based single-cell proteomic profiling reveals heterogeneity in DNA damage response dynamics

doi: 10.1016/j.crmeth.2022.100237

Figure Lengend Snippet: Single-cell quantitative proteomics on group 1 and group 2 cells collected by the FUNpro pipeline (A) Phototagging of the group 2 cells. Scale bar: 500 μm. The zoomed-in images showed a representative cell (arrow) before and after phototagging. Scale bar: 50 μm. Red, 53BP1; green, phototagging (PT) dye. (B) Schematic protocol of single-cell proteomics analysis. Four non-tagged cells, four tagged cells, and 200 non-tagged cells (serving as carrier cells) were labeled with respective 10-plex TMT labels as indicated and then mixed into one sample before being subjected to liquid chromatography tandem mass spectrometry (LC-MS/MS). Ten samples, in total 40 group 1 and 40 group 2 cells, were analyzed. (C) (i) A volcano plot showing proteins enriched either in group 1 (normal DDR) or group 2 (abnormal DDR) cells. Together, 80 cells were pooled into the analysis. Dashed lines show the cutoff value of FDR at 0.05. (ii) DDR-related protein, PDS5A, was found upregulated in group 2 cells.

Article Snippet: The cells were then incubated with 1:500 rabbit polyclonal anti-PDS5A antibody (Novus) or 1:1000 rabbit polyclonal anti-γH2AX antibody (Abcam) for 90 min at room temperature.

Techniques: Labeling, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy

Journal: Cell Reports Methods

Article Title: Microscopy-based single-cell proteomic profiling reveals heterogeneity in DNA damage response dynamics

doi: 10.1016/j.crmeth.2022.100237

Figure Lengend Snippet: Immunofluorescence validation of the PDS5A protein on group 1 and group 2 (with asterisk) cells (A) (i) A UFO image of U2OS cells expressing 53BP1-mScarlet (gray). The image was taken after 24 h of IR. Scale bar: 10 μm. (ii) A confocal image of immunofluorescence against the PDS5A protein (16 colored) from the same fieldof-view. Scale bar: 10 μm. (B) Immunofluorescence quantification of the PDS5A protein on group 1 and group 2 cells. In total, 16 pairs of cells were analyzed with p value lower than 0.01 (Student’s t test).

Article Snippet: The cells were then incubated with 1:500 rabbit polyclonal anti-PDS5A antibody (Novus) or 1:1000 rabbit polyclonal anti-γH2AX antibody (Abcam) for 90 min at room temperature.

Techniques: Immunofluorescence, Expressing

Journal: Cell Reports Methods

Article Title: Microscopy-based single-cell proteomic profiling reveals heterogeneity in DNA damage response dynamics

doi: 10.1016/j.crmeth.2022.100237

Figure Lengend Snippet:

Article Snippet: The cells were then incubated with 1:500 rabbit polyclonal anti-PDS5A antibody (Novus) or 1:1000 rabbit polyclonal anti-γH2AX antibody (Abcam) for 90 min at room temperature.

Techniques: Recombinant, Transfection, Software, Cell Tracking Assay, Mass Spectrometry

Journal: Cell Reports Methods

Article Title: Microscopy-based single-cell proteomic profiling reveals heterogeneity in DNA damage response dynamics

doi: 10.1016/j.crmeth.2022.100237

Figure Lengend Snippet: Single-cell quantitative proteomics on group 1 and group 2 cells collected by the FUNpro pipeline (A) Phototagging of the group 2 cells. Scale bar: 500 μm. The zoomed-in images showed a representative cell (arrow) before and after phototagging. Scale bar: 50 μm. Red, 53BP1; green, phototagging (PT) dye. (B) Schematic protocol of single-cell proteomics analysis. Four non-tagged cells, four tagged cells, and 200 non-tagged cells (serving as carrier cells) were labeled with respective 10-plex TMT labels as indicated and then mixed into one sample before being subjected to liquid chromatography tandem mass spectrometry (LC-MS/MS). Ten samples, in total 40 group 1 and 40 group 2 cells, were analyzed. (C) (i) A volcano plot showing proteins enriched either in group 1 (normal DDR) or group 2 (abnormal DDR) cells. Together, 80 cells were pooled into the analysis. Dashed lines show the cutoff value of FDR at 0.05. (ii) DDR-related protein, PDS5A, was found upregulated in group 2 cells.

Article Snippet: The cells were then incubated with 1:500 rabbit polyclonal anti-PDS5A antibody (Novus) or 1:1000 rabbit polyclonal anti-γH2AX antibody (Abcam) for 90 min at room temperature.

Techniques: Quantitative Proteomics, Labeling, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy

Journal: Cell Reports Methods

Article Title: Microscopy-based single-cell proteomic profiling reveals heterogeneity in DNA damage response dynamics

doi: 10.1016/j.crmeth.2022.100237

Figure Lengend Snippet: Immunofluorescence validation of the PDS5A protein on group 1 and group 2 (with asterisk) cells (A) (i) A UFO image of U2OS cells expressing 53BP1-mScarlet (gray). The image was taken after 24 h of IR. Scale bar: 10 μm. (ii) A confocal image of immunofluorescence against the PDS5A protein (16 colored) from the same fieldof-view. Scale bar: 10 μm. (B) Immunofluorescence quantification of the PDS5A protein on group 1 and group 2 cells. In total, 16 pairs of cells were analyzed with p value lower than 0.01 (Student’s t test).

Article Snippet: The cells were then incubated with 1:500 rabbit polyclonal anti-PDS5A antibody (Novus) or 1:1000 rabbit polyclonal anti-γH2AX antibody (Abcam) for 90 min at room temperature.

Techniques: Immunofluorescence, Biomarker Discovery, Expressing

Journal: Cell Reports Methods

Article Title: Microscopy-based single-cell proteomic profiling reveals heterogeneity in DNA damage response dynamics

doi: 10.1016/j.crmeth.2022.100237

Figure Lengend Snippet:

Article Snippet: The cells were then incubated with 1:500 rabbit polyclonal anti-PDS5A antibody (Novus) or 1:1000 rabbit polyclonal anti-γH2AX antibody (Abcam) for 90 min at room temperature.

Techniques: Recombinant, Transfection, Software, Cell Tracking Assay, Mass Spectrometry

Journal: Cell Reports Methods

Article Title: Microscopy-based single-cell proteomic profiling reveals heterogeneity in DNA damage response dynamics

doi: 10.1016/j.crmeth.2022.100237

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-PDS5A , Novus , Cat#NBP1-87904; RRID: AB_11015600.

Techniques: Recombinant, Transfection, Software, Cell Tracking Assay, Mass Spectrometry