Journal: Journal of Cellular and Molecular Medicine

Article Title: Ailanthone synergizes with PARP1 inhibitor in tumour growth inhibition through crosstalk of DNA repair pathways in gastric cancer

doi: 10.1111/jcmm.18033

Figure Lengend Snippet: AIL with OLP blocks HSP90‐BRCA1 and PARP1 activity. The protein expression of HSP90, BRCA1, PARP1 in two GC cell lines after treated with a blank control group, OLP, AIL and OLP + AIL group. (B) The protein expression of HSP90, BRCA1 in two GC cell lines after being treated with a specified concentration of AIL for 24 h. (C) Pull‐down of selected proteins with HSP90 in the nucleus and cytoplasm of GC cells treated with AIL for 24 h, and then the IP fractions were immunoblotted with HSP90, BRCA1 and GAPDH. (D) Representative images of HSP90, BRCA1 and PARP1 in PDX tumour tissue as detected via immunohistochemistry. Scale bars, 200 μm. (E) Data were derived from experiments conducted in triplicate in (D). The PDX tumour tissues were compared with the control group, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: BRCA1 (1:100, Proteintech, 22362‐1‐AP).

Techniques: Activity Assay, Expressing, Control, Concentration Assay, Immunohistochemistry, Derivative Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: Ailanthone synergizes with PARP1 inhibitor in tumour growth inhibition through crosstalk of DNA repair pathways in gastric cancer

doi: 10.1111/jcmm.18033

Figure Lengend Snippet: AIL inhibits BRCA1 through downregulating P23 and inevitably suppress the HR pathway of GC. Western blotting was performed to detect the expression of P23, HSP90 and BRCA1 in AGS and SGC7901 cells after treatment with a specified concentration of CEL (P23 inhibitor) for 24 h. (B) The mRNA expression of P23 in two GC cell lines (AGS and SGC7901) after being treated with P23 shRNA lentivirus. (C) The protein expression of P23, HSP90 and BRCA1 in two GC cell lines after being treated with shP23‐1, shP23‐2 and shP23‐3. (D‐F) BALB/C nude mice are implanted with shNC or shP23‐3 GC cells subcutaneously. (D) The images of subcutaneous tumour nodules formed in two groups of mice. (E) Representative images of P23, HSP90 and BRCA1 in CDX tumour tissue as detected via immunohistochemistry. Scale bars, 50 μm. (F) Data were derived from experiments conducted in triplicate in (E).

Article Snippet: BRCA1 (1:100, Proteintech, 22362‐1‐AP).

Techniques: Western Blot, Expressing, Concentration Assay, shRNA, Immunohistochemistry, Derivative Assay

Journal: Molecular Neurobiology

Article Title: Oxymatrine Alleviates Cerebral Ischemia/Reperfusion Injury By Targeting HDAC1 to Regulate Mitochondria-Related Autophagy and Oxidative Stress

doi: 10.1007/s12035-025-05423-1

Figure Lengend Snippet: Oxymatrine ameliorates excessive autophagy induced by cerebral I/R in mice. A Western blotting results. B – G Western blotting analyses of LC3-II/I, PINK1, Parkin, NBR1, Beclin-1 and P62 in different groups when normalized to GAPDH. H – K Representative images showing double immunofluorescence staining for autophagy-related proteins (LC3, PINK1, Beclin1, NBR1) co-localized with neurons in different groups. Scale bar = 20 μm. # p < 0.05 vs. Sham, * p < 0.05 vs. I/R

Article Snippet: Protein extracts were separated on 10%–12% SDS-PAGE gels, transferred to PVDF membranes (Millipore), and probed with the following primary antibodies: Apaf-1 (1:1000, ABclonal, Wuhan, China), cleaved-caspase3 (1:500, Proteintech, Wuhan, China), Fis1 (1:2000, Proteintech, Wuhan, China), NBR1 (1:1000, Proteintech, Wuhan, China), Mfn2 (1:4000, Proteintech, Wuhan, China), LC3 (1:1000, ABclonal, Wuhan, China), HDAC1(1:2000, ABclonal, Wuhan, China), PINK1 (1:800, ABclonal, Wuhan, China), Parkin (1:2000, Proteintech, Wuhan, China), Beclin1 (1:2000, Proteintech, Wuhan, China), p62 (1:2000,Proteintech, Wuhan, China).

Techniques: Western Blot, Double Immunofluorescence Staining

Journal: Molecular Neurobiology

Article Title: Oxymatrine Alleviates Cerebral Ischemia/Reperfusion Injury By Targeting HDAC1 to Regulate Mitochondria-Related Autophagy and Oxidative Stress

doi: 10.1007/s12035-025-05423-1

Figure Lengend Snippet: Oxymatrine restores mitochondrial homeostasis via coordinated modulation of fission/fusion balance and autophagy in glutamate-treated HT22 cells. A Western blotting for Fis1 and Mfn2. B , C Quantification of western blotting. D Immunofluorescence analysis showing the expression of LC3, PINK1, Beclin-1, NBR1 and P62. Scale bar = 20 μm. E Western blotting for LC3-II/I, PINK1, Beclin-1, NBR1 and P62. F – K Quantification of western blotting. # p < 0.05 vs. Control, * p < 0.05 vs. Glu

Article Snippet: Protein extracts were separated on 10%–12% SDS-PAGE gels, transferred to PVDF membranes (Millipore), and probed with the following primary antibodies: Apaf-1 (1:1000, ABclonal, Wuhan, China), cleaved-caspase3 (1:500, Proteintech, Wuhan, China), Fis1 (1:2000, Proteintech, Wuhan, China), NBR1 (1:1000, Proteintech, Wuhan, China), Mfn2 (1:4000, Proteintech, Wuhan, China), LC3 (1:1000, ABclonal, Wuhan, China), HDAC1(1:2000, ABclonal, Wuhan, China), PINK1 (1:800, ABclonal, Wuhan, China), Parkin (1:2000, Proteintech, Wuhan, China), Beclin1 (1:2000, Proteintech, Wuhan, China), p62 (1:2000,Proteintech, Wuhan, China).

Techniques: Western Blot, Immunofluorescence, Expressing, Control

Journal: Molecular Biology Reports

Article Title: Involvement of a coumarin analog AD-013 in the DNA damage response pathways in MCF-7 cells

doi: 10.1007/s11033-018-4271-z

Figure Lengend Snippet: A Real-time PCR analysis of ATM, ATR, DNA-PK, BRCA1, p53, TBP, ABL-1, Chk1 and Chk2 mRNA levels in MCF-7 cells treated with AD-013. Data are expressed as mean ± SEM. Statistical significance was assessed by the Student t -test. * p < 0.05 and ** p < 0.01 was considered as significantly different from control. B Effect of AD-013 on phosphorylation of ATM Ser1981 in MCF-7 cells. Protein extracts obtained after treatment of cells with the tested compound (at IC 50 and 2×IC 50 concentrations) were assayed for ATM phosphorylation (activation), using the RayBio Phospho-ATM (Ser1981) and Total ATM protein Kit. Data are expressed as mean ± SEM. Statistical significance was assessed using one-way ANOVA and a post-hoc multiple comparison Student–Newman–Keuls test. *** p < 0.001, in comparison with control. C Influence of AD-013 on BRCA1 protein concentration in MCF-7 cells. Extracts from MCF-7 cells treated with AD-013 at IC 50 concentration were assayed for BRCA1 activation, using the Human BRCA1 ELISA kit. Data are presented as mean ± SEM. Statistical significance was assessed by the Student t test * p < 0.05, in comparison with control

Article Snippet: Determination of BRCA1 protein level in MCF-7 cells incubated with AD-013 was analyzed by Human BRCA1 ELISA kit (Elabscience, Houston, TX, USA).

Techniques: Real-time Polymerase Chain Reaction, Control, Phospho-proteomics, Activation Assay, Comparison, Protein Concentration, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Signal Transduction and Targeted Therapy

Article Title: PKMYT1 kinase ameliorates cisplatin sensitivity in osteosarcoma

doi: 10.1038/s41392-025-02250-7

Figure Lengend Snippet: PKMYT1-induced NPM1 S260 phosphorylation promotes efficient DSB repair. a Western blot analysis of the expression of PKMYT1, phosphorylated-NPM1 S260, and phosphorylated-NPM1 T199 in OS cells after being treated with various concentrations of DDP. b – d Western blot analysis of the effect of DDP and IR on NPM1 S260 phosphorylation after PKMYT1 knockdown or inhibition with RP6306. e Western blot analysis of the impact of DDP and IR on NPM1 S260 phosphorylation following the overexpression of NPM1 S260 dephosphorylation mutant plasmid. f After transfection as indicated siRNAs for 48 h, HEK293T cells were collected and subjected to co-immunoprecipitation experiments followed by western blot analysis. g HEK293T cells were transfected with indicated siRNAs for 48 h and treated with or without cisplatin (1 μM) for 24 h before harvesting the cells for co-immunoprecipitation and Western blot. h HEK293T cells were treated with or without RP6306 (2 μM) for 24 h before harvesting the cells for co-immunoprecipitation and Western blot. i HEK293T cells were treated with or without RP6306 (2 μM) and cisplatin (1 μM) for 24 h before harvesting the cells for co-immunoprecipitation and Western blot. j HEK293T cells were transfected with indicated plasmids for 24 h and treated with or without cisplatin (1 μM) before harvesting the cells for co-immunoprecipitation and Western blot. k – l Immunofluorescence analysis of the effect of NPM1 S260 phosphorylation on IR-induced BRCA1 foci. NPM1 knockout U2OS cells were rescued with either wild-type NPM1, NPM1 S260A mutant and NPM1 S260D mutant, with or without IR (10 Gy) treatment. Representative images are shown in k , and the number of BRCA1 foci per group was calculated in l. Approximately 100 cells were counted per group. Data are expressed as the mean ± SEM from three biological replicates. Statistical analysis was performed using Student’s t test, p value as indicated. Scale bar, 10 μm. m – n Immunofluorescence analysis of PKMYT1 wild-type and PKMYT1 knockout U2OS cells with or without IR (10 Gy) treatment. Representative immunofluorescence images were shown in m, and the number of BRCA1 foci per group was calculated in n, p value as indicated. Scale bar, 10 μm. o – p NPM1 knockout U2OS cells were rescued with indicated plasmids for 24 h, and immunofluorescence analysis of cells exposed to 10 Gy IR and recovered for 1 h. Representative immunofluorescence images were shown in o , and the number of RAD51 foci per group was shown in p , p value as indicated. Scale bar, 10 μm. q – r Immunofluorescence analysis of PKMYT1 wild-type and PKMYT1 knockout U2OS cells with or without IR (10 Gy) treatment. Representative immunofluorescence images were shown in ( q ), and the number of RAD51 foci per group was calculated in r . Scale bar, 10 μm. s , t Immunofluorescence analysis of PKMYT1 wild-type and PKMYT1 knockout cells with or without IR (10 Gy) treatment. Representative immunofluorescence images were shown in ( s ), and the number of RAP80 foci per group was calculated in t , p value as indicated. Scale bar, 10 μm. u HR assay in PKMYT1 wild-type and PKMYT1 knockout HEK293T cells, with BRCA1 knockout as control. Data are presented as the mean ± SEM from three replicates. Each group included over 1000 cell counts. P value as indicated. v NHEJ assay in PKMYT1 wild-type and PKMYT1 knockout HEK293T cells, with 53BP1 knockout as control. Data are presented as the mean ± SEM from three replicates. Each group included over 1000 cell counts, p value as indicated

Article Snippet: The antibodies used in this study are as follows: GAPDH (#60004-1-Ig, Proteintech, 1:10,000 dilution), PKMYT1 (#4282S, Cell Signaling Technology, 1:1,000 dilution), NPM1 (#60096-1-Ig, Proteintech, 1:5,000 dilution), p-NPM1 T199 (#AF3111, Affnity, 1:1000 dilution), p-NPM1 S260 (#TP50488, HUABIO, 1:3000 dilution), p-Histone H2A.X S139 (# P40705 , ProMab Biotechnologies Inc., China, 1:1000 dilution), RAP80 (#13642-1-AP, Proteintech, 1:1000 dilution), BRCA1 (#22362-1-AP, Proteintech, 1:1000 dilution), RAD51(#14961-1-AP, Proteintech, China, 1:2000 dilution), anti-HA (#AB0004, Abways, China, 1:2000 dilution), β-actin (A00730, Genscript Biotech, 1:1000 dilution), Phosphoserine/threonine/tyrosine (#11995 R, Yajikit, 1:100 dilution), HRP conjugated goat-anti-mouse antibody (#SA00001-1, Proteintech, 1:10,000 dilution) and HRP conjugated goat-anti-rabbit antibody (#511203, zen-bioscience, 1:5000 dilution).

Techniques: Phospho-proteomics, Western Blot, Expressing, Knockdown, Inhibition, Over Expression, De-Phosphorylation Assay, Mutagenesis, Plasmid Preparation, Transfection, Immunoprecipitation, Immunofluorescence, Knock-Out, Control, NHEJ Assay

Journal: Diagnostic Pathology

Article Title: The ectopic expression of BRCA1 is associated with genesis, progression, and prognosis of breast cancer in young patients

doi: 10.1186/1746-1596-7-181

Figure Lengend Snippet: A (young group) BRCA1 protein immunohistochemical staining was positive. The positive products localized in the cytoplasm (× 400). B (young group) BRCA1 protein immunohistochemical staining was positive. The positive products localized in the nucleus (× 400). C (old group) BRCA1 protein immunohistochemical staining was positive. The positive products localized in the cytoplasm (× 400). D (young group) WWOX protein immunohistochemical staining was positive. The positive products localized in the cytoplasm (× 400). E (old group) WWOX protein immunohistochemical staining was positive. The positive products localized in the cytoplasm (× 400) . F (young group) ER protein immunohistochemical staining was positive. The positive product positioning in the nucleus (× 400). G (young group) positive immunohistochemical staining of PR proteins. The positive products localized in the nucleus (× 400) H (young group) C-erbB2 protein immunohistochemical staining was positive. The positive products localized in the membrane (× 400). I (young group) Ki67 protein immunohistochemical staining was positive. The positive products localized in the nucleus (× 400).

Article Snippet: The WWOX antibody was purchased from Beijing Boaosen Biotechnology Co., Ltd. BRCA1 antibody was purchased from Wuhan Boster Biological Engineering Co., Ltd. ER, PR, and Ki67 were expressed in the nucleus.

Techniques: Immunohistochemical staining, Staining, Membrane

Journal: Diagnostic Pathology

Article Title: The ectopic expression of BRCA1 is associated with genesis, progression, and prognosis of breast cancer in young patients

doi: 10.1186/1746-1596-7-181

Figure Lengend Snippet: Analysis of the correlation between the BRCA1 results and clinicopathological parameters in the young groups and old groups

Article Snippet: The WWOX antibody was purchased from Beijing Boaosen Biotechnology Co., Ltd. BRCA1 antibody was purchased from Wuhan Boster Biological Engineering Co., Ltd. ER, PR, and Ki67 were expressed in the nucleus.

Techniques:

Journal: Diagnostic Pathology

Article Title: The ectopic expression of BRCA1 is associated with genesis, progression, and prognosis of breast cancer in young patients

doi: 10.1186/1746-1596-7-181

Figure Lengend Snippet: A The PCR product of exon 2 of the BRCA1 gene (259 bp). B The PCR product of exon 20 of the BRCA1 gene (401 bp).

Article Snippet: The WWOX antibody was purchased from Beijing Boaosen Biotechnology Co., Ltd. BRCA1 antibody was purchased from Wuhan Boster Biological Engineering Co., Ltd. ER, PR, and Ki67 were expressed in the nucleus.

Techniques:

Journal: Diagnostic Pathology

Article Title: The ectopic expression of BRCA1 is associated with genesis, progression, and prognosis of breast cancer in young patients

doi: 10.1186/1746-1596-7-181

Figure Lengend Snippet: A The partial DNA sequencing results of exon 2 of BRCA1 gene of patient 6. B The partial DNA sequencing results of exon 20 of BRCA1 gene of patient 6.

Article Snippet: The WWOX antibody was purchased from Beijing Boaosen Biotechnology Co., Ltd. BRCA1 antibody was purchased from Wuhan Boster Biological Engineering Co., Ltd. ER, PR, and Ki67 were expressed in the nucleus.

Techniques: DNA Sequencing

Journal: Diagnostic Pathology

Article Title: The ectopic expression of BRCA1 is associated with genesis, progression, and prognosis of breast cancer in young patients

doi: 10.1186/1746-1596-7-181

Figure Lengend Snippet: Comparative analysis of the immunohistochemical results of young and old patient groups

Article Snippet: The WWOX antibody was purchased from Beijing Boaosen Biotechnology Co., Ltd. BRCA1 antibody was purchased from Wuhan Boster Biological Engineering Co., Ltd. ER, PR, and Ki67 were expressed in the nucleus.

Techniques: Immunohistochemical staining

Journal: Nutrients

Article Title: Epigenetic Activation of BRCA1 by Genistein In Vivo and Triple Negative Breast Cancer Cells Linked to Antagonism toward Aryl Hydrocarbon Receptor

doi: 10.3390/nu11112559

Figure Lengend Snippet: Lifetime exposure to genistein (GEN) decreases basal Brca1 cytosine-guanine dinucleotide (CpG) methylation and aryl hydrocarbon receptor (AHR) activity in mouse offspring mammary tissue. Mice were exposed to diets containing 0, 4, or 10 ppm GEN starting at conception and through nursing, weaning, and adulthood. ( A ) Brca1 CpG methylation in a mammary gland of post-natal day 50 (PND50) female offspring. Bars represent the mean ratios of methylated Brca1 to unmethylated Brca1 amplicons ± SEM from ≥7 individual animals. ( B ) Cyp1b1 mRNA expression in PND50 mammary glands. Bars represent mean RQ ± SEM from ≥7 individual animals. Means with different letters (a > b > c) or asterisks (**, p < 0.01) indicate statistical significance ( p < 0.05).

Article Snippet: Immunoblotting was carried out with antibodies raised against BRCA1 (Boster Bio, Pleasanton, CA, USA; Ref. PB9015), ERα (Santa Cruz, Ref. MC-20), AHR (Santa Cruz Biotechnology Inc, Dallas, TX, USA; Ref. B-11), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Origene, Rockville, MD, USA; Ref. TA890003).

Techniques: CpG Methylation Assay, Activity Assay, Methylation, Expressing

Journal: Nutrients

Article Title: Epigenetic Activation of BRCA1 by Genistein In Vivo and Triple Negative Breast Cancer Cells Linked to Antagonism toward Aryl Hydrocarbon Receptor

doi: 10.3390/nu11112559

Figure Lengend Snippet: BRCA1 CpG hypermethylation associates with constitutively active AHR in triple negative breast cancers (TNBC) cells. ( A ) Representative Western blot images comparing immunocomplexes of AHR and internal standard glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in HCC38 and UACC3199 cells. ( B ) Data from methylation-specific PCR (MSP) comparing BRCA1 and ESR1 CpG methylation in HCC38 and MCF7 cells. ( C ) Bands represent immunocomplexes for BRCA1, estrogen receptor (ER)α, AHR, and internal standard GAPDH in MCF7 and HCC38 cells. ( D ) Comparison of AHR mRNA expression in HCC38 and MCF7 cells. ( E ) Expression of AHR targets CYP1A1 and CYP1B1 in HCC38 and MCF7 cells. ( F ) CYP1A1 and CYP1B1 expression in HCC38 cells treated with the AHR antagonist CH-223191. Bars represent sample means ± SEM from ≥3 biological replicates from individual experiments. VEH: vehicle-treated control. Asterisks indicate significant differences (*, p < 0.05; **, p < 0.01).

Article Snippet: Immunoblotting was carried out with antibodies raised against BRCA1 (Boster Bio, Pleasanton, CA, USA; Ref. PB9015), ERα (Santa Cruz, Ref. MC-20), AHR (Santa Cruz Biotechnology Inc, Dallas, TX, USA; Ref. B-11), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Origene, Rockville, MD, USA; Ref. TA890003).

Techniques: Western Blot, Methylation, CpG Methylation Assay, Comparison, Expressing, Control

Journal: Nutrients

Article Title: Epigenetic Activation of BRCA1 by Genistein In Vivo and Triple Negative Breast Cancer Cells Linked to Antagonism toward Aryl Hydrocarbon Receptor

doi: 10.3390/nu11112559

Figure Lengend Snippet: Genistein rescues BRCA1 protein levels in TNBC cells with CpG hypermethylated BRCA1 . Bands are immunocomplexes of BRCA1 and internal standard GAPDH in HCC38 cells treated with ( A ) GEN, ( B ) α-naphthoflavone (NF), ( C ) CH-223191, and ( D ) galangin (GAL). Images are representative blots from ≥3 individual experiments. For densitometry histograms, bars represent mean RQ ± SEM from ≥3 biological replicates from individual experiments. Asterisks indicate significant differences (*, p < 0.05; ***, p < 0.001).

Article Snippet: Immunoblotting was carried out with antibodies raised against BRCA1 (Boster Bio, Pleasanton, CA, USA; Ref. PB9015), ERα (Santa Cruz, Ref. MC-20), AHR (Santa Cruz Biotechnology Inc, Dallas, TX, USA; Ref. B-11), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Origene, Rockville, MD, USA; Ref. TA890003).

Techniques:

Journal: Nutrients

Article Title: Epigenetic Activation of BRCA1 by Genistein In Vivo and Triple Negative Breast Cancer Cells Linked to Antagonism toward Aryl Hydrocarbon Receptor

doi: 10.3390/nu11112559

Figure Lengend Snippet: Rescue of BRCA1 expression is linked to CpG demethylation in TNBC cells. The average ratio of mBRCA1 / umBRCA1 in HCC38 cells treated for 96 h with ( A ) GEN or ( B ) GAL. Bars represent sample means ± SEM from ≥3 biological replicates from individual experiments. Asterisks denote significance (*, p < 0.05).

Article Snippet: Immunoblotting was carried out with antibodies raised against BRCA1 (Boster Bio, Pleasanton, CA, USA; Ref. PB9015), ERα (Santa Cruz, Ref. MC-20), AHR (Santa Cruz Biotechnology Inc, Dallas, TX, USA; Ref. B-11), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Origene, Rockville, MD, USA; Ref. TA890003).

Techniques: Expressing

Journal: Nutrients

Article Title: Epigenetic Activation of BRCA1 by Genistein In Vivo and Triple Negative Breast Cancer Cells Linked to Antagonism toward Aryl Hydrocarbon Receptor

doi: 10.3390/nu11112559

Figure Lengend Snippet: Antagonism toward AHR binding at the BRCA1 promoter by GEN. Results show the relative binding of AHR at the BRCA1 promoter from chromatin immunoprecipitation (ChIP) assays in ( A ) HCC38 cells treated with GEN and various AHR antagonists; and ( B ) MCF7 cells treated with estradiol (E2) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the presence or absence of GEN and GAL. Relative binding was determined as the percent input (ChIP/Input*100) from treated samples normalized to VEH ( A ) or E2 ( B ) controls. Bars represent sample means ± SEM from ≥3 biological replicates from individual experiments. Means with different letters (a > b) or asterisks (*) indicate significance (*, p < 0.05).

Article Snippet: Immunoblotting was carried out with antibodies raised against BRCA1 (Boster Bio, Pleasanton, CA, USA; Ref. PB9015), ERα (Santa Cruz, Ref. MC-20), AHR (Santa Cruz Biotechnology Inc, Dallas, TX, USA; Ref. B-11), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Origene, Rockville, MD, USA; Ref. TA890003).

Techniques: Binding Assay, Chromatin Immunoprecipitation

Journal: Molecular and cellular endocrinology

Article Title: AKT Regulates BRCA1 Stability in Response to Hormone Signaling

doi: 10.1016/j.mce.2010.01.019

Figure Lengend Snippet: (A) MCF7 and MDA-MB-231 cells were cultured for either 48 hours in 10% charcoal stripped serum (CSS) or 24 hours in serum starved (0.5%) conditions. Control cells were cultured in normal growth medium (10% fetal bovine serum). Cell lysates (100 μg) were immunoblotted for BRCA1, pS473 AKT, and pan AKT. pS473 AKT indicates activation of AKT; pan AKT serves as a loading control. Molecular weights (kD) are indicated. The upper band in the BRCA1 panel for MCF7 cells is the full length (p220) BRCA1 protein. The lower band may represent a splice variant that is not consistently observed across experimental replicates or conditions.

Article Snippet: Purified full length BRCA1 protein was obtained from ProteinOne. pGEX-BRCA1 S694/T696 fusion protein was phosphorylated by AKT1 and prepared for mass spectrometry as previously described ( 69 ).

Techniques: Cell Culture, Control, Activation Assay, Variant Assay

Journal: Molecular and cellular endocrinology

Article Title: AKT Regulates BRCA1 Stability in Response to Hormone Signaling

doi: 10.1016/j.mce.2010.01.019

Figure Lengend Snippet: (A) MCF7 cells cultured in normal growth medium were treated with the indicated doses of LY294002 for 18 hours and then harvested for immunoblot analysis. Controls include both untreated cells and DMSO vehicle treated cells. Molecular weights (kD) are indicated. The immunoblot was probed with rabbit polyclonal phospho-AKT-substrate antibody (PAS), then stripped and reprobed with a mouse monoclonal BRCA1 antibody. An immunoreactive band was observed (*) of the same molecular weight with both antibodies which decreased in a dose-dependent manner upon LY294002 treatment. Immunoblots for BARD1, pS473 AKT, and pan AKT are presented in the third panel. The pS473 AKT antibody bound to a cross-reactive band of lower molecular weight in this experiment which was not recognized by the pan AKT antibody.

Article Snippet: Purified full length BRCA1 protein was obtained from ProteinOne. pGEX-BRCA1 S694/T696 fusion protein was phosphorylated by AKT1 and prepared for mass spectrometry as previously described ( 69 ).

Techniques: Cell Culture, Western Blot, Molecular Weight

Journal: Molecular and cellular endocrinology

Article Title: AKT Regulates BRCA1 Stability in Response to Hormone Signaling

doi: 10.1016/j.mce.2010.01.019

Figure Lengend Snippet: (A) Full length, purified BRCA1 protein (250 ng/lane) was added to an in vitro kinase assay plus or minus active AKT1 kinase. GST and GST-GSK3 were included as negative and positive controls respectively. Molecular weights (kD) are indicated. Reaction products were separated by SDS-PAGE and 32P-labelled products were detected by exposure to a phosphor screen. Silver stain demonstrated equal loading of BRCA1 (*) in the experimental lanes. The predominant band in all lanes is BSA (a component of the enzyme dilution buffer).

Article Snippet: Purified full length BRCA1 protein was obtained from ProteinOne. pGEX-BRCA1 S694/T696 fusion protein was phosphorylated by AKT1 and prepared for mass spectrometry as previously described ( 69 ).

Techniques: Purification, In Vitro, Kinase Assay, SDS Page, Silver Staining

Journal: Molecular and cellular endocrinology

Article Title: AKT Regulates BRCA1 Stability in Response to Hormone Signaling

doi: 10.1016/j.mce.2010.01.019

Figure Lengend Snippet: (A) Whole cell extracts were prepared from MCF7 cells and separated by SDS-PAGE. After transfer, the PVDF membrane was split longitudinally and each lane was incubated separately with the indicated antibodies: mouse monoclonal anti-BRCA1 (Ab4) and rabbit polyclonal anti-pS694 BRCA1 (at dilutions of 1:500 and 1:1000). Additionally, pS694 BRCA1 antibody at a dilution of 1:500 was incubated with the membrane either without or with pre-incubation with the specific phosphorylated antigen matrix (lanes four and five respectively) as a control for specificity of the antibody. Separately incubated lanes (1-3 and 4-5) were reassembled and exposed to the same piece of film for chemiluminescent detection. Molecular weights (kD) are indicated.

Article Snippet: Purified full length BRCA1 protein was obtained from ProteinOne. pGEX-BRCA1 S694/T696 fusion protein was phosphorylated by AKT1 and prepared for mass spectrometry as previously described ( 69 ).

Techniques: SDS Page, Membrane, Incubation, Control

Journal: Molecular and cellular endocrinology

Article Title: AKT Regulates BRCA1 Stability in Response to Hormone Signaling

doi: 10.1016/j.mce.2010.01.019

Figure Lengend Snippet: (A) MCF7 cells cultured in 10% CSS medium were stimulated with 10 nM E2 or pretreated with 10 μM LY294002 for 1 hour before stimulation with E2 for indicated amount of time (in hours). Immunoblot for BRCA1 and BARD1 proteins demonstrates that pre-incubation with LY294002 blocks the increase in total protein levels observed with E2 treatment alone. Samples treated with LY294002 alone showed similar levels of BRCA1 and BARD1 expression as the E2 + LY294002 groups (data not shown). pS473-AKT was detected to monitor AKT activation. Numbers below each panel represent relative densitometry values compared to the vehicle control.

Article Snippet: Purified full length BRCA1 protein was obtained from ProteinOne. pGEX-BRCA1 S694/T696 fusion protein was phosphorylated by AKT1 and prepared for mass spectrometry as previously described ( 69 ).

Techniques: Cell Culture, Western Blot, Incubation, Expressing, Activation Assay, Control

Journal: Molecular and cellular endocrinology

Article Title: AKT Regulates BRCA1 Stability in Response to Hormone Signaling

doi: 10.1016/j.mce.2010.01.019

Figure Lengend Snippet: (A) MCF7 cells were transduced with the indicated adenoviral vectors, cultured for 48 hours in CSS medium, then fixed and stained with both an N-terminal mouse monoclonal BRCA1 antibody (red) and an exon 11 directed rabbit polyclonal BRCA1 antibody (green). Yellow indicates overlap of both signals to demonstrate specific staining for BRCA1. Nuclei were counterstained with DAPI. The following MOI's were used: 100 for Ad-myr-AKT, Ad-kd-AKT, and control Ad-LacZ; 25 for Ad-BRCA1.

Article Snippet: Purified full length BRCA1 protein was obtained from ProteinOne. pGEX-BRCA1 S694/T696 fusion protein was phosphorylated by AKT1 and prepared for mass spectrometry as previously described ( 69 ).

Techniques: Transduction, Cell Culture, Staining, Control

Journal: Molecular and cellular endocrinology

Article Title: AKT Regulates BRCA1 Stability in Response to Hormone Signaling

doi: 10.1016/j.mce.2010.01.019

Figure Lengend Snippet: MCF7 cells were transduced with the indicated adenoviral vectors and plated at low density in triplicate in six-well plates. Cells were cultured in CSS medium for 24 hours prior to irradiation with indicated doses and maintained in CSS medium for an additional 48 hours following irradiation. Cells were then switched into normal growth medium to allow for colony outgrowth. Survival is presented on a logarithmic scale as the number of remaining colonies at each dose divided by the average number of colonies in unirradiated wells of the same transduction group. The following MOI's were used: 100 for Ad-myr-AKT, Ad-kd-AKT, and control Ad-GFP; 10 for Ad-BRCA1 and Ad-BR1853. Error bars represent SEM, N=3. A student's t-test was performed for BRCA1/myr-AKT vs. mock, GFP, myr-AKT, BRCA1/GFP, or BR1853/myr-AKT and found to be significant at p≤ 0.01 for both 2 and 4 Gy treatment groups (*) in all cases.

Article Snippet: Purified full length BRCA1 protein was obtained from ProteinOne. pGEX-BRCA1 S694/T696 fusion protein was phosphorylated by AKT1 and prepared for mass spectrometry as previously described ( 69 ).

Techniques: Transduction, Cell Culture, Irradiation, Control