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A B C
IBMDM Control RAW264.7
S.T - + + S.T S.T - + +
GSK8612 - - + 1.5 S.T+GSK8612 GSK8612 - - +
kDa ** kDa
Supernatant 20 Caspase⁃1⁃P20 1.0 * ** Supernatant 20 Caspase⁃1⁃P20
53 Caspase⁃1 Relative protein expression level 53 Caspase⁃1
53 GSDMD 0.5 53 GSDMD
Lysis Lysis
35 GSDMD⁃N 0 35 GSDMD⁃N
80 p⁃TBK1 80 p⁃TBK1
Caspase⁃1⁃P20
35 GAPDH GSDMD⁃N p⁃TBK1 35 GAPDH

D E F
IBMDM
Control * GSK8612 - - + 1.5 Control
Relative protein expression level 0.8 * * Supernatant 20 Caspase⁃1⁃P20 Relative protein expression level 1.0 *** *** ***
FLIC - + +
FLIC
S.T
S.T+GSK8612
FLIC+GSK8612
1.0
kDa
Caspase⁃1
53
0.6
53
GSDMD
0.4
0.5
Lysis
0.2
GSDMD⁃N
35
0
0
Caspase⁃1⁃P20 GSDMD⁃N p⁃TBK1 80 p⁃TBK1 Caspase⁃1⁃P20 GSDMD⁃N p⁃TBK1
35
GAPDH
G H I
IBMDM IBMDM RAW264.7
*** *** ***
0.8 *** * 0.6 *** *** 1.0 *** ***
（mU/mL） 0.6 （mU/mL） 0.4 （mU/mL） 0.8
0.6
0.4
LDH 0.2 LDH 0.2 LDH 0.4
0.2
0 0 0
S.T
Control S.T+GSK8612 Control FLIC+GSK8612 Control S.T+GSK8612
S.T
FLIC
J K L
RAW264.7 Control RAW264.7
S.T - + + S.T 0.8 ***
shTBK1 - - + S.T+shTBK1 *** ***
kDa 1.0 * *** *** 0.6
Supernatant 20 Caspase⁃1⁃P20 0.8 ** （mU/mL） 0.4
53 Caspase⁃1 0.6
53 GSDMD Relative protein expression level 0.4 LDH 0.2
Lysis 35 GSDMD⁃N 0.2 0 S.T
80 p⁃TBK1 0 Control S.T+shTBK1
Caspase⁃1⁃P20
80 TBK1 p⁃TBK1 TBK1
35 GAPDH GSDMD⁃N
After two hours of pre⁃treatment with GSK8612，using S.T（MOI：50）stimulated IBMDM（A，B）or RAW264.7（C，D）for 2 h，or activated NLRC4
inflammasomes by transfecting flagellin（FLIC）into IBMDM（E，F），and Western blot was used to detect expression levels of related proteins in cell
lysate and culture supernatant samples. Simultaneously，a lactate dehydrogenase（LDH）detection kit was used to detect the secretion of LDH in cell
culture supernatant（G，H，I）. After knockdowning TBK1 in RAW264.7 cells，expression levels of related proteins in cell lysate and cell culture superna⁃
**
*
tant samples were detected（J，K），and the secretion of LDH in cell culture supernatant was detected（L）. P < 0.05，P < 0.01，and *** P < 0.001（n=3）.
图1 TBK1参与调控NLRC4炎症小体激活
Figure 1 TBK1 regulates the activation of NLRC4 inflammasome

［11］
化对于NLRC4炎症小体的激活至关重要 ，因此本 HEK293T细胞中过量表达Flag⁃TBK1和Myc⁃NLRC4
研究检测磷酸化酶 TBK1 能否磷酸化 NLRC4。在 或 Myc⁃NLRC4（Ser533A）（NLRC4 的 永 久 非 磷 酸