anti mdc1 rrid ab 323725  (Bio-Rad)

Journal: eLife

Article Title: ARID1A governs the silencing of sex-linked transcription during male meiosis in the mouse

doi: 10.7554/eLife.88024

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Article Snippet: Antibody , anti-MDC1 RRID: AB_323725 (Sheep polyclonal) , AbD Serotec , AHP799 , IF (1:500).

Techniques:

Journal: Nature Communications

Article Title: Non-canonical function of DGCR8 in DNA double-strand break repair signaling and tumor radioresistance

doi: 10.1038/s41467-021-24298-z

Figure Lengend Snippet: a Immunoblotting of Drosha, DGCR8, and β-actin in the LM2-DRR (expressing the pLCN DSB Repair Reporter) cell line transduced with DGCR8 shRNA. b Knockdown of DGCR8 decreased HR and NHEJ efficiency in LM2-DRR cells. Two days after co-transfection of I-SceI endonuclease and an exogenous donor for HR (pCAGGS DRR mCherry Donor EF1a BFP) into the DGCR8-knockdown LM2-DRR cells, the percentages of GFP-positive and mCherry-positive cells, gated on BFP-positive cells, were determined by flow cytometry. Repair by HR or NHEJ leads to mCherry or GFP expression. Data were normalized to the control cells. n = 3 biological replicates. c MYC-DGCR8-overexpressing LM2 cells were treated with IR (8 Gy) and cultured for 1 h, followed by pulldown with MYC beads and immunoblotting with the indicated antibodies. d Control and DGCR8-knockdown LM2 cells were treated with IR (8 Gy) and cultured for 1 h, followed by immunoprecipitation with an antibody against RNF168 or RNF8 and immunoblotting with the indicated antibodies. e Chromatin was extracted from LM2 cells that were treated with IR (8 Gy) and cultured for 1 h. The chromatin fractions, with or without MNase treatment, were immunoprecipitated with a DGCR8-specific antibody and immunoblotted with the indicated antibodies. f Quantification of MDC1, RNF8, RNF168, 53BP1, and BRCA1 foci in DGCR8-knockdown LM2 cells. Cells were incubated for 1 h after 2-Gy IR and immunostained with antibodies against γH2AX, MDC1, RNF8, RNF168, 53BP1, and BRCA1 (see representative images in Supplementary Fig. ). n = 3 biological replicates. g Control and DGCR8-knockdown LM2 cells with stable overexpression of FLAG-H2A and RNF8 or RNF168 were transfected with HA-ubiquitin (Ub), treated with IR (8 Gy), and cultured for 8 h, followed by immunoprecipitation with anti-FLAG beads and immunoblotting with antibodies against HA and FLAG. Before immunoprecipitation, lysates were heated at 95 °C for 5 min in the presence of 1% SDS (for denaturing), followed by a 10-fold dilution with lysis buffer and sonication. LE long exposure, SE short exposure. Statistical significance in b and f was determined by a two-tailed unpaired t -test. Error bars are mean ± SEM. n.s . not statistically significant. Source data are provided as a file.

Article Snippet: The cells were washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS, blocked with 3% bovine serum albumin in PBS, and incubated with antibodies against γH2AX (1:100, Cell Signaling Technology, #9718S), γH2AX (1:100, BD Biosciences, #560443), DGCR8 (1:100, Abcam, #ab191875), MDC1 (1:200, Bio-Rad, #AHP799), RNF8 (1:200, Proteintech, #14112-1-AP), RNF168 (1:100, Millipore, #ABE367), BRCA1 (1:20, Santa Cruz Biotechnology, #sc-6954), 53BP1 (1:100, Novus Biologicals, #NB100-304), FLAG (1:500, Sigma, #F7425), and MYC (1:500, Santa Cruz Biotechnology, #sc-40, clone 9E10) at 4 °C overnight, followed by incubation with Alexa Fluor 488 goat anti-rabbit IgG (1:1000, Invitrogen, ThermoFisher Scientific, #A-11008), Alexa Fluor 647 donkey anti-sheep IgG (1:1000, Invitrogen, ThermoFisher Scientific, #A-21448), Alexa Fluor 488 goat anti-mouse IgG (1:1000, Invitrogen, ThermoFisher Scientific, #A-11001), Alexa Fluor 594 goat anti-rabbit IgG (1:1000, Invitrogen, ThermoFisher Scientific, #A-11012), and/or Alexa Fluor 594 goat anti-mouse IgG (1:1000, Invitrogen, ThermoFisher Scientific, #A-11005) at room temperature for 1 h. Coverslips were mounted on slides by using anti-fade mounting medium with 4′,6-diamidino-2-phenylindole (DAPI, Vector Laboratories, #H-1200).

Techniques: Western Blot, Expressing, Transduction, shRNA, Knockdown, Cotransfection, Flow Cytometry, Control, Cell Culture, Immunoprecipitation, Incubation, Over Expression, Transfection, Ubiquitin Proteomics, Lysis, Sonication, Two Tailed Test

Journal: Nature Communications

Article Title: Non-canonical function of DGCR8 in DNA double-strand break repair signaling and tumor radioresistance

doi: 10.1038/s41467-021-24298-z

Figure Lengend Snippet: a Immunoblotting of USP36, USP51, and β-actin in parental and radioresistant LM2 cells with and without IR treatment (8 Gy followed by 24-h incubation). b Immunoblotting of DGCR8, USP36, USP51, and β-actin in USP36-knockdown and USP51-knockdown LM2 cells with or without IR treatment (8 Gy followed by 24-h incubation). c Co-IP of endogenous DGCR8 with endogenous USP51. LM2 and LM2-R cells were treated with 8-Gy IR. After 8 h, cells were lysed, immunoprecipitated with a DGCR8-specific antibody, and immunoblotted with antibodies against USP51 and DGCR8. SE short exposure, LE long exposure. d HEK293T cells with stable overexpression of MYC-DGCR8 were co-transfected with SFB-USP51 (wild-type or the C372S mutant) and HA-tagged ubiquitin or the lysine-specific mutant (K48 or K63), and then treated with IR (8 Gy). After 8 h, cells were lysed, denatured, and subjected to immunoprecipitation with anti-MYC beads and immunoblotting with antibodies against HA and MYC. e Knockdown of USP51 decreased HR and NHEJ efficiency in LM2-DRR cells. Two days after co-transfection of I-SceI endonuclease and an exogenous donor for HR (pCAGGS DRR mCherry Donor EF1a BFP) into the USP51-knockdown LM2-DRR cells, the percentages of GFP-positive and mCherry-positive cells, gated on BFP-positive cells, were determined by flow cytometry. Repair by HR or NHEJ leads to mCherry or GFP expression. Data were normalized to the control cells. n = 3 biological replicates. f Quantification of γH2AX, DGCR8, MDC1, RNF8, RNF168, 53BP1, and BRCA1 foci in USP51-knockdown LM2 cells. Cells were incubated for 1 h after 2-Gy IR and immunostained with antibodies against γH2AX, DGCR8, MDC1, RNF8, RNF168, 53BP1, and BRCA1 (see representative images in Supplementary Fig. ). n = 3 biological replicates. g Control and USP51-knockdown LM2 cells with stable overexpression of FLAG-H2A and RNF8 or RNF168 were transfected with HA-ubiquitin (Ub), treated with IR (8 Gy), and cultured for 8 h, followed by immunoprecipitation with anti-FLAG beads and immunoblotting with antibodies against HA and FLAG. Before immunoprecipitation, lysates were heated at 95 °C for 5 min in the presence of 1% SDS (for denaturing), followed by a 10-fold dilution with lysis buffer and sonication. Statistical significance in e and f was determined by a two-tailed unpaired t -test. Error bars are mean ± SEM. Source data are provided as a file.

Article Snippet: The cells were washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS, blocked with 3% bovine serum albumin in PBS, and incubated with antibodies against γH2AX (1:100, Cell Signaling Technology, #9718S), γH2AX (1:100, BD Biosciences, #560443), DGCR8 (1:100, Abcam, #ab191875), MDC1 (1:200, Bio-Rad, #AHP799), RNF8 (1:200, Proteintech, #14112-1-AP), RNF168 (1:100, Millipore, #ABE367), BRCA1 (1:20, Santa Cruz Biotechnology, #sc-6954), 53BP1 (1:100, Novus Biologicals, #NB100-304), FLAG (1:500, Sigma, #F7425), and MYC (1:500, Santa Cruz Biotechnology, #sc-40, clone 9E10) at 4 °C overnight, followed by incubation with Alexa Fluor 488 goat anti-rabbit IgG (1:1000, Invitrogen, ThermoFisher Scientific, #A-11008), Alexa Fluor 647 donkey anti-sheep IgG (1:1000, Invitrogen, ThermoFisher Scientific, #A-21448), Alexa Fluor 488 goat anti-mouse IgG (1:1000, Invitrogen, ThermoFisher Scientific, #A-11001), Alexa Fluor 594 goat anti-rabbit IgG (1:1000, Invitrogen, ThermoFisher Scientific, #A-11012), and/or Alexa Fluor 594 goat anti-mouse IgG (1:1000, Invitrogen, ThermoFisher Scientific, #A-11005) at room temperature for 1 h. Coverslips were mounted on slides by using anti-fade mounting medium with 4′,6-diamidino-2-phenylindole (DAPI, Vector Laboratories, #H-1200).

Techniques: Western Blot, Incubation, Knockdown, Co-Immunoprecipitation Assay, Immunoprecipitation, Over Expression, Transfection, Mutagenesis, Ubiquitin Proteomics, Cotransfection, Flow Cytometry, Expressing, Control, Cell Culture, Lysis, Sonication, Two Tailed Test

Journal: Nature Communications

Article Title: Non-canonical function of DGCR8 in DNA double-strand break repair signaling and tumor radioresistance

doi: 10.1038/s41467-021-24298-z

Figure Lengend Snippet: a , b MYC-GFP-, WT DGCR8-, S677A-DGCR8-, and S677D-DGCR8-overexpressing LM2 cells with or without IR treatment ( a , 8 Gy followed by 1-h incubation; b , 8 Gy followed by 8-h incubation) were subjected to pulldown with MYC beads and immunoblotting with the indicated antibodies. c HEK293T cells with stable overexpression of MYC-tagged WT DGCR8, S677A-DGCR8, or S677D-DGCR8 were co-transfected with SFB-USP51 (WT or the C372S mutant) and HA-tagged ubiquitin, and then treated with IR (8 Gy). After 8 h, cells were lysed, denatured, and subjected to immunoprecipitation with anti-MYC beads and immunoblotting with antibodies against HA and MYC. d Quantification of γH2AX, DGCR8, MDC1, RNF8, RNF168, 53BP1, and BRCA1 foci in DRCR8-knockdown LM2 cells with ectopic expression of WT DGCR8, S677A-DGCR8, or S677D-DGCR8. Cells were incubated for 1 h after 2-Gy IR and immunostained with antibodies against γH2AX, DGCR8, MDC1, RNF8, RNF168, 53BP1, and BRCA1 (see representative images in Supplementary Fig. ). n = 3 biological replicates. Statistical significance was determined by a two-tailed unpaired t -test. Error bars are mean ± SEM. e DRCR8-knockdown LM2 cells with ectopic expression of WT DGCR8 or the S677A mutant were transduced with FLAG-H2A and RNF8 or RNF168. The cells were then transfected with HA-ubiquitin (Ub), treated with IR (8 Gy), and cultured for 8 h, followed by immunoprecipitation with anti-FLAG beads and immunoblotting with antibodies against HA and FLAG. Before immunoprecipitation, lysates were heated at 95 °C for 5 min in the presence of 1% SDS (for denaturing), followed by a 10-fold dilution with lysis buffer and sonication. LE long exposure, SE short exposure. Source data are provided as a file.

Article Snippet: The cells were washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS, blocked with 3% bovine serum albumin in PBS, and incubated with antibodies against γH2AX (1:100, Cell Signaling Technology, #9718S), γH2AX (1:100, BD Biosciences, #560443), DGCR8 (1:100, Abcam, #ab191875), MDC1 (1:200, Bio-Rad, #AHP799), RNF8 (1:200, Proteintech, #14112-1-AP), RNF168 (1:100, Millipore, #ABE367), BRCA1 (1:20, Santa Cruz Biotechnology, #sc-6954), 53BP1 (1:100, Novus Biologicals, #NB100-304), FLAG (1:500, Sigma, #F7425), and MYC (1:500, Santa Cruz Biotechnology, #sc-40, clone 9E10) at 4 °C overnight, followed by incubation with Alexa Fluor 488 goat anti-rabbit IgG (1:1000, Invitrogen, ThermoFisher Scientific, #A-11008), Alexa Fluor 647 donkey anti-sheep IgG (1:1000, Invitrogen, ThermoFisher Scientific, #A-21448), Alexa Fluor 488 goat anti-mouse IgG (1:1000, Invitrogen, ThermoFisher Scientific, #A-11001), Alexa Fluor 594 goat anti-rabbit IgG (1:1000, Invitrogen, ThermoFisher Scientific, #A-11012), and/or Alexa Fluor 594 goat anti-mouse IgG (1:1000, Invitrogen, ThermoFisher Scientific, #A-11005) at room temperature for 1 h. Coverslips were mounted on slides by using anti-fade mounting medium with 4′,6-diamidino-2-phenylindole (DAPI, Vector Laboratories, #H-1200).

Techniques: Incubation, Western Blot, Over Expression, Transfection, Mutagenesis, Ubiquitin Proteomics, Immunoprecipitation, Knockdown, Expressing, Two Tailed Test, Transduction, Cell Culture, Lysis, Sonication