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실험계획. hTERT cell line 에 PAH 노출 후 molecular study (cell count, Viability, Copy number variation 등 ) Leukemia cell line - THP-1 cell line 에서 발굴된 Protein { Prelamin -A/C(LMNA), Poly [ADP-ribose] polymerase 1(PARP-1)} 의 mRNA 수준에서 expression study
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실험계획 hTERT cell line에 PAH 노출 후 molecular study (cell count, Viability, Copy number variation등) Leukemia cell line - THP-1 cell line 에서 발굴된 Protein {Prelamin-A/C(LMNA), Poly [ADP-ribose] polymerase 1(PARP-1)}의 mRNA 수준에서 expression study - Western blotting을 이용한 LMNA, PARP-1 Protein 분석: mitochondria fraction, cytosolic fraction - Immunofluorescence stain을 이용하여 PARP-1과 LMNA가 localization되어 있는 위치 확인 3. In vivo Zebrafish model - Western blotting을 이용한 Protein 분석: 시약주문 완료 - Protein (LMNA, PARP-1)의 mRNA 수준에서 expression study SETBP1 mutation in MDS/MPN Patients
Amplification plot and melting curve analysis: PARP-1, LMNA, b-actin
mRNA expression: LMNA exon1 (Normalized with b-actin gene) 1day zebrafish embryos
mRNA expression: LMNA exon1 (Normalized with b-actin gene) 2day zebrafish embryos
mRNA expression: PARP-1 exon4 (Normalized with b-actin gene) 1day zebrafish embryos
mRNA expression: PARP-1 exon4 (Normalized with b-actin gene) 2day zebrafish embryos
Immunofluorescence stain Slide glass에 target cell cytospin (600rpm, 5min) 4% paraformaldehyed/PBS, 10min, RT에서 고정 PBS washing, 5min, 3times PBS-T(0.1% Tween20), 15min incubation Primary antibody (PARP-1) 1:100 in PBST, 4°c O/N PBS-T(1% BSA 첨가) washing, 5min, 3times Secondary antibody (mouse Green 488) 1:500 in PBST, 2~3hrs Mitotracker Red CMXRos200nM 처리 PBS-T(1% BSA 첨가) washing, 5min, 3times PBS:DAPI=10,000:1, 10min PBS-T(1% BSA 첨가) washing, 5min, 2times PBS washing, 5min, 1times
cover glass에 target cell cytospin (600rpm, 5min) 4% paraformaldehyed/PBS, 10min, RT에서 고정 PBS washing, 5min, 3times PBS-T(0.1% Tween20), 15min incubation Primary antibody (PARP-1) 1:50 in PBST, 4°c O/N PBS-T(1% BSA 첨가) washing, 5min, 3times Secondary antibody (mouse Green 488) 1:100 in PBST, 2~3hrs Mitotracker Red CMXRos 1:100 (10nM) 처리 PBS-T(1% BSA 첨가) washing, 5min, 3times PBS:DAPI=10,000:1, 10min PBS-T(1% BSA 첨가) washing, 5min, 2times PBS washing, 5min, 1times
Trouble shooting: Mitotracker Red의 적정 희석배수와염색시간 찾기 – • 10nM, 1nM, 0.1nM, 0.01nM에서 10분, 30분 염색 testing
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 607bp M 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
G2898 (Asp868) G2898G/A (Asp868Asp/Asn) Patient No.7 Patient 9 Patient No.10
C2903 (ser869) Patient No.13 C2903T/C (ser869)