Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Inflammasome priming by LPS is dependent upon ERK signaling and proteasome function *

doi: 10.4049/jimmunol.1301974

Figure Lengend Snippet: Monocytes were nucleofected with ERK1 and ERK2 specific siRNA or scrambled control and recovered overnight. Cells were then primed with LPS (1μg/ml) for 30 min and activated with ATP (5mM) for another 30 min. Supernatants were analyzed for IL-18 release and cell lysates were immunoblotted to confirm ERK protein down regulation using total ERK antibody. IL-18 ELISA represents the mean± SEM for three separate monocyte donors and the blots are representative of repeated blots. **p < 0.01 as compared to scrambled siRNA.

Article Snippet: In knockdown experiments, small interfering RNA (siRNA) against ERK1 and scrambled siRNA were purchased from Sigma-Aldrich while Signalsilencer ® p42 MAPK (ERK2) siRNA II was purchased from Cell Signaling Company. siRNAs were delivered in monocytes by Amaxa nucleofector I (Lonza).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Journal of Inflammation Research

Article Title: CCL20 Expression via AKT-ERK1/2-AP1 Pathway in Mycoplasma Pneumoniae Infection: Implications for EMT and Cell Migration

doi: 10.2147/JIR.S512408

Figure Lengend Snippet: The ERK1/2 signaling pathway is involved in regulating the expression of CCL20. THP-1 cells were infected with M. pneumoniae at MOIs of 0, 10, 50, or 100 for 1 h, and protein expression levels were assessed by Western blot ( A and B ). In inhibitor experiments, cells were pretreated with 30 µM ERK1/2 inhibitor PD98059 for 1 h before infection with M. pneumoniae . The inhibitory effect was detected by Western blot ( C and D ). The levels of CCL20 mRNA and protein were measured by RT-qPCR ( E ) and ELISA ( F ), respectively. Additionally, cells were pretreated with 50 µM LY294002 prior to infection, and the inhibitory effect on ERK1/2 was detected by Western blot ( G and H ). Representative results from three independent experiments are shown. * p <0.05, ** p <0.01, **** p <0.0001. * indicates significance compared to the control group.

Article Snippet: Subsequently, the membrane was incubated overnight at 4°C with primary antibodies, including HY-P80276 P-AKT, HY-P80009 AKT, HY-P80813 P-ERK1/2, HY-P80393 ERK1/2, HY-P80084 c-jun, HY-P80616 c-fos, and HY-P80112 E-cadherin (all from MCE, USA, diluted at 1:800), as well as HY-P80438 β-actin (from MCE, USA, diluted at 1:10,000) and D155001 p-c-jun, D151325 p-c-fos, and D121239 snail (from Sangon Biotech, China, diluted at 1:800).

Techniques: Expressing, Infection, Western Blot, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Beta-PIX and Rac1 GTPase mediate trafficking and negative regulation of NOD2.

doi: 10.4049/jimmunol.181.4.2664

Figure Lengend Snippet: FIGURE 2. Effect of Rac1 siRNA and the Rac1 inhibitor NSC23766 on NOD2- and TLR2-mediated IL-8 secretion. Primary human monocytes were transfected with control nonsilencing siRNA (c-siRNA) or siRNA targeting Rac1 (si-Rac1). After 72 h, cells were lysed and Western blots using anti-Rac1 Abs were performed (A). Membranes were simultaneously probed with anti-ERK2 Abs to confirm equal protein loading. B, Primary human monocytes were transfected with siRNAs as indicated, and after 72 h, stimulated with MDP or Malp2 for 16 h, and the supernatants were analyzed for IL-8 secretion by ELISA. THP-1 cells were untreated (none) or preincubated overnight with the Rac1 inhibitor NSC23766 (200 M). Subsequently, the cells were either stimulated with MDP (C) or Malp2 (D) for 16 h, and the supernatants were analyzed for IL-8 secretion by ELISA. Data presented are mean SD of three different experiments performed in duplicates (, p 0.01).

Article Snippet: Membranes were exposed to Abs specific to Rac1 (Transduction Laboratories), NOD2 (ProSci), Nalp3 (Biozol), -Pix, c-Myc, Erbin, or ERK2 (Santa Cruz Biotechnology), respectively.

Techniques: Transfection, Control, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Beta-PIX and Rac1 GTPase mediate trafficking and negative regulation of NOD2.

doi: 10.4049/jimmunol.181.4.2664

Figure Lengend Snippet: FIGURE 3. Influence of Rac1 on NOD2- or TLR2-mediated IL-8 and NF-B activation. A, HEK293 cells were left untreated, or were trans- fected with control non-silencing siRNA (c-siRNA) or siRNA targeting Rac1 (si-Rac1). After 72 h, cells were lysed and Western blots using anti-Rac1 Abs were performed in duplicates. Membranes were simul- taneously probed with anti-ERK2 Abs to confirm equal protein loading. B–D, HEK293 cells were left untreated (ctrl), or were transfected with control non-silencing siRNA (c-siRNA) or siRNA targeting Rac1 (si- Rac1). After 48 h, the cells were additionally cotransfected with NOD2 (B and C) or TLR2 (D) expression plasmids, together with an IL-8- reporter (B) or NF-B reporter construct (C and D) and a -galactosi- dase reporter plasmid. Cells were either left untreated () or stimulated with MDP (B and C) or Malp2 (D) and relative luciferase activities were obtained. E and F, HEK293 cells seeded in 24-well plates were tran- siently transfected with a control vector (ctrl) or NOD2 expression plas- mid along with an IL-8 luciferase reporter plasmid (E) or a NF-B- driven luciferase reporter (F), respectively, and a -galactosidase reporter plasmid. Additionally, the cells were cotransfected with wild- type Rac1 (Rac1wt), dominant negative Rac1N17 or constitutively ac- tive Rac1L61. Cells were either stimulated with 10 g/ml MDP (MDP) or left untreated (), and relative luciferase activities were obtained the next day. G, HEK293 cells seeded in 24-well plates were transiently transfected with a control vector (ctrl), or a RIP2 expression plasmid along with a NF-B-driven luciferase reporter and a -galactosidase plasmid. The influence of Rac1 was tested by additionally introducing wild-type Rac1 (Rac1wt), dominant negative Rac1 (Rac1N17), or con- stitutive active Rac1 (Rac1L61) mean SD; , p 0.01.

Article Snippet: Membranes were exposed to Abs specific to Rac1 (Transduction Laboratories), NOD2 (ProSci), Nalp3 (Biozol), -Pix, c-Myc, Erbin, or ERK2 (Santa Cruz Biotechnology), respectively.

Techniques: Activation Assay, Control, Western Blot, Transfection, Expressing, Construct, Plasmid Preparation, Luciferase, Dominant Negative Mutation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Beta-PIX and Rac1 GTPase mediate trafficking and negative regulation of NOD2.

doi: 10.4049/jimmunol.181.4.2664

Figure Lengend Snippet: FIGURE 7. Rac1 and -PIX siRNAs as well as the Rac1 inhibitor NSC23766 inhibit interaction of NOD2 with Erbin. Primary monocytes (A and B) or THP-1 cells (C) were either preincubated with the Rac1 inhibitor NSC23766 (NSC) or were transfected with Rac1 siRNA or -PIX siRNA, as indicated, and were stimulated with 10 g/ml MDP (MDP) for 40 min. Subsequently, immunoprecipitations with an Erbin Ab and subsequent im- munoblots with NOD2 and ERK2 Abs (A) or NOD2, Rac1, and Erbin Abs (B and C) were performed. One representative Western blot out of three is shown.

Article Snippet: Membranes were exposed to Abs specific to Rac1 (Transduction Laboratories), NOD2 (ProSci), Nalp3 (Biozol), -Pix, c-Myc, Erbin, or ERK2 (Santa Cruz Biotechnology), respectively.

Techniques: Transfection, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Beta-PIX and Rac1 GTPase mediate trafficking and negative regulation of NOD2.

doi: 10.4049/jimmunol.181.4.2664

Figure Lengend Snippet: FIGURE 6. Involvement of -PIX in NOD2-medi- ated signaling. MDP-stimulated primary monocytes or THP-1 cells (A and B) were lysed at different time points, immunoprecipitations of endogenous Rac1 (A) or endogenous NOD2 (B) with the respective Abs were performed, and immune complexes were probed for the presence of -Pix. Equal protein amounts in the lysates were confirmed by blotting total cell lysates with an ERK2 Ab (Input). All experiments were repeated three times. C, THP-1 cells were transfected with control non-silencing siRNA (c-siRNA) or siRNA targeting -Pix (si--Pix_S1 (sequence 1), si-Pix_S2 (sequence 2)). After 72 h, cells were lysed and Western blots using anti--Pix Abs were performed. Western blots were si- multaneously probed with anti-ERK2 Abs to confirm equal protein loading. D, THP-1 cells were transfected as indicated, incubated for 72 h, and stimulated with MDP (10 g/ml) for 40 min. Membrane and cytosol fractions were separated and immunoblotted with anti- NOD2 Abs. E and F, THP-1 cells were transfected with siRNAs as indicated, and after 72 h, stimulated with MDP (E) or Malp2 (F) for 16 h, and the supernatants were analyzed for IL-8 secretion by ELISA. Data pre- sented are mean SD of three different experiments performed in duplicates (, p 0.01).

Article Snippet: Membranes were exposed to Abs specific to Rac1 (Transduction Laboratories), NOD2 (ProSci), Nalp3 (Biozol), -Pix, c-Myc, Erbin, or ERK2 (Santa Cruz Biotechnology), respectively.

Techniques: Transfection, Control, Sequencing, Western Blot, Incubation, Membrane, Enzyme-linked Immunosorbent Assay