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STAT3 Undergoes Acetylation‐dependent Mitochondrial Translocation to Regulate Pyruvate Metabolism
Yan S. Xu1, Jinyuan J. Liang1, Yumei Wang2, Xiang‐zhong J. Zhao1,2, Li
a b
, y g , g , g g , ,Xu3, Ye‐yang Xu1, Quanli C. Zou2, Junxun M. Zhang2, Cheng‐e Tu2, Yan‐geCui2, Wei‐hong Sun2, Chao Huang1,4, Jing‐hua Yang1,*, Y. Eugene Chin2,*
Seq # b y +1--- -- ------ ------ --I 1 114.1 - 8 K 2 242.2 906.5 7 Q 3 370.2 778.4 6 F 4 517.3 650.3 5 L 5 630.4 503.3 4 Q 6 758.4 390.2 3 S 7 845.5 262.1 2 R 8 - 175.1 1
peptide=IKQFLQSR, (510.3 d)
Seq # b y +1--- -- ------ ------ --I 1 114.1 - 8 *K 2 284.2 948.5 7 Q 3 412.2 778.4 6 F 4 559.3 650.3 5 L 5 672.4 503.3 4 Q 6 800.5 390.2 3 S 7 887.5 262.1 2 R 8 - 175.1 1
peptide=IKQFLQSR, (531.3 d)
Anti‐aK87 AB
Anti‐aK707 AB
Anti‐aK709 AB
K87
K707
K709
c
IP:
293Ts
‐Flag‐STAT3IB: Flag
‐Flag‐SIRT5IB: Flag
IP:
‐Myc‐STAT3IB: Myc
‐Flag‐SIRT5IB: Flag
293Ts
IP:‐INSRIB: INSR
‐CBPIB: CBP
MEFs
IP:‐STAT3 IB:STAT3
‐aK87 IB:aK87
SS/R
‐aK‐STAT3 IB: aK (pan)
IP:‐Myc‐STAT3
IB: Myc‐Myc‐PDC E1
IB: Myc293Ts
IP:
‐Flag‐STAT3IB: Flag
‐Myc‐PDC E1IB: Myc293Ts
MEFs
IP:
MEFs
‐STAT3IB: STAT3‐PDC E1IB: PDC E1
‐aK685 IB: aK685
‐aK707 IB: aK707
‐aK709 IB: aK709
‐pY705 IB: pY705
‐pS727 IB: pS727
MEFs
Figure S1. (a) Peptide slot blot analysis of the antibodies prepared for STAT3 acetylation on K87, K707 and K709. (b) y and b numbers of the mass spectrum in Fig. 2b. (c) IgG controls were detedted for Immunoprecipitationand co‐immunoprecipitation experiments involved in this text.
a b c CBP‐HA: + +
INSR: + +INS: ‐ +
‐INSR STAT3
STAT3:‐STAT3
STAT3: ‐ + +CBP: ‐ ‐ +
1 6 1 23 4
d e
‐CBP‐HA
INSR
‐CBP‐HA
‐INSR Input
IP:CBP 1 1.7STAT3
‐Porin
Mit
1 49.4 44.3 36 31
‐STAT3
‐Porin
‐aK87
‐Tubulin
Mit
Cyt
1 6.1 23.4
1 29.0 54.9
d e
‐STAT3
STAT3:
Cyt Mit
‐Porin
‐Tubulin
1 1.3 0.3 0.6‐aK‐STAT3
‐STAT3
‐Porin
(pan)1 1.2 2.4
1 12.2 27.7
Figure S2. (a) INSR and CBP were overexpressed in 293T cells. HA‐CBP was immunoprecipitated followed by Western blotting analysis with anti‐INSR and anti‐HA. The bottom panel gave the input of INSR and HA‐CBP. (b) 293T ll t f t d ith EV STAT3 STAT3 d CBP STAT3
Porin
Mit
293T cells were transfected with EV, STAT3, or STAT3 and CBP. STAT3 acetylation on lysine 87 was detected with a specific antibody via Western blotting. (c) 293T cells were transfected with EV, wild‐type STAT3 and STAT3 with the N‐terminus, C‐terminus, or both N‐ and C‐termini deleted (i.e., 1‐738, 28‐770, and 28‐738). Mitochondria were isolated from the 293T transfectants and STAT3 and Porin levels were shown by Western293T transfectants and STAT3 and Porin levels were shown by Western blotting. (d) STAT3‐K87R mutant or STAT3‐WT was transiently transfectedinto 293T cells. Cytoplasmic and mitochondrial fractions were prepared for STAT3 localization analysis via Western blotting with anti‐Myc. (e)Mitochondrial lysates were prepared for STAT3 acetylation analysis from 293T cells transfected with EV, STAT3 or Cox4‐STAT3.
Fig. 1a
170170130
Figure S3. Full scans of western blotting data in all figures.