. 근육 분화 A, 면역 침전 (IP) 동안 IRS-1/2 및 근생 신호 분자의 인산화 상태의 변화. 표시된 기간에 세포 용해는 항 IRS-1/2 항체에 의해 면역침전되었다. 면역침전액은 IRS-1/2, 포스포세린, 인포스포티로신(PY20) 및 p85에 특이적인 항체를 사용하여 웨스턴 블롯(IB) 분석에 의해 분석되었다. IRS-1/2 면역침전제에 대한 PI 3-키나아제 활성은 재료 및 방법에 기재된 바와 같이 측정하였다. PIP는 인산화 지질을 나타낸다. B, 웨스턴 블롯 분석. 세포 용해액은 Ser307 및 Ser636/639 및 그 발현 수준에서 IRS-1 인산화 수준에 대해 분석되었다. C 와 D, IRS-1/2 단백질 양에 샘플의 정상화. IRS-1/2에 대한 시료 양을 정상화하기 위해, PM에 있는 세포용액보다 DM에서 3배 더 높은 양의 세포 용해액을 항 IRS-1/2 항체(C) 또는 웨스턴 블롯 분석(D)에 의해 면역침전을 행하였다. 액틴 수준은 로드된 샘플 양을 나타냅니다.
이들 모든 실험은 적어도 3회 이상 반복되었고 전형적인 데이터가 제시된다. PI 3-키나아제 아세술은 앞서 설명한 바와 같이 수행되었다(17). PI 3-키나아제 활성은 PI를 포스파티딜리노시톨포스페이트(PIP)로 전환시킴으로써 IRS-1 또는 2 면역침전제에서 분석하였다. 세포는 용해 완충액 1 ml에서 30분 동안 인큐베이션하여 용해시켰다[20 mm Tris-HCl (pH 7.5), 137 mm NaCl, 1 mm MgCl2, 1 mm CaCl2, 1mm EDTA, 1% 노니데트 P-40, 10% 글리세롤, 5 μg/1 mms.1 mm. 항-IRS-1 또는 -2 항체 응액 아가로즈(20 μl)를 4C에서 1시간 동안 500 μg의 단백질을 함유하는 슈퍼나이트로 배양하였다. 세척 후, 면역침전액을 키나아제 분석 버퍼[20 mm Tris-HCl (pH 7.6), 75 mm NaCl, 10 mm MgCl2, 200 μg/ml PI 및 10 μCi [γ-32P] ATP]의 100 μl에서 재중단시키고, 실온에서 20분 동안 배양하였다. 반응을 1n HCl 100 μl 및 CHCl3-메탄올 200 μl(1:1)의 첨가에 의해 중단하였다. 샘플을 원심분리하고, 하부 유기상을 수확하고 실리카 겔 박층 크로마토그래피 플레이트(Merck, Whitehouse Station, NJ)에 1% 칼륨 옥살레이트를 코팅하였다. 얇은 층 크로마토그래피 플레이트는 CHCl3-CH3OH-H2O-NH4OH (60:47:11.3:2)에서 개발되었으며, 건조되고 자가 방사선 촬영에 의해 시각화되었습니다. 추가적으로, DM에서FGF-2는 SER307 인산화를 증가시켰지만, IRS-1의 티로신 인산화를 감소시켰으며, 이는 PM에서 관찰된 바와 같이 IRS-1 및 PI 3-키나아제 활성과 p85의 감소된 연관성에 뒤따랐다.
이들 분자에 대한 FGF-2의 효과는 4d에 대한 DM에서 Y27632 및 FGF-2의 공동 처리에 의해 역전되었다(도 8D).
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