Journal: Molecular and cellular neurosciences

Article Title: Differential role of Dok1 and Dok2 in TLR2-induced inflammatory signaling in glia.

doi: 10.1016/j.mcn.2013.04.007

Figure Lengend Snippet: Fig. 3. Dok1 and Dok2 are adaptors associated with TLR2 signaling to ERK and NF-κB in glia. Primary mouse (A) astrocytes and (B) microglia were transfected with non-targeting siRNA and ON-TARGET plus siRNA targeting mouse Dok1 and Dok2. After 72 h, cell lysates were prepared and subjected to western immunoblotting using anti-Dok1, anti-Dok2 and anti-β-actin antibodies. Primary (C) astrocytes and (D) microglia were transfected with non-targeting siRNA and Dok1/Dok2 siRNA. After 72 h, cells were treated with Pam3CSK4 (100 ng/ml; 10–60 min), lysates were prepared and subjected to immunoblotting using anti-phospho-ERK, anti-ERK2 and anti-β-actin antibodies. (E) Astrocytes and (F) microglia were transfected with non-targeting siRNA and Dok1/Dok2 siRNA. After 72 h, cells were treated with Pam3CSK4 (100 ng/ml; 20 min), and cytosolic fractions were subjected to western immunoblotting using anti-phospho-IκB-α and anti-β-actin antibodies. Nuclear fractions were prepared and assessed for NF-κB activity by gel mobility shift assay. All data are mean ± SEM and are representative of data obtained from 12 animals. ⁎p b 0.05 and ⁎⁎⁎p b 0.001 compared with vehicle-treated cells (A, B, E, F) and untreated control siRNA-transfected cells (C, D). +++p b 0.001 compared with untreated Dok1 siRNA-transfected cells (D) and non-transfected cells treated with Pam3CSK4 (E, F). ##p b 0.01 and ###p b 0.001 compared with untreated Dok2 siRNA-transfected cells (C, D).

Article Snippet: Membranes were incubated overnight at 4 °C with mouse monoclonal phospho-ERK antibody (1:1000 in 5% dried milk; Santa Cruz Biotechnology, Santa Cruz, CA), mouse monoclonal phospho-IκB-α antibody (1:1000 in 5% dried milk; Cell Signaling Technology Inc., Danvers, MA) or rabbit polyclonal Dok1 and Dok2 antibodies (1:500 in 5% BSA; Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Transfection, Western Blot, Activity Assay, Mobility Shift, Control

Journal: Molecular and cellular neurosciences

Article Title: Differential role of Dok1 and Dok2 in TLR2-induced inflammatory signaling in glia.

doi: 10.1016/j.mcn.2013.04.007

Figure Lengend Snippet: Fig. 5. Schematic representation of the mechanism by which Dok1 and Dok2 mediate TLR2-induced signaling in astrocytes and microglia. (A) In astrocytes, both Dok1 and Dok2 act as classic negative regulators of TLR2-induced ERK phosphorylation, NF-κB activation and pro-inflammatory cytokine expression. (B) In microglia, both Dok1 and Dok2 act as negative regulators of TLR2-induced ERK phosphorylation. However, Dok1, but not Dok2, promotes IκB-α phosphorylation, NF-κB DNA binding activity and pro-inflammatory cytokine production. Pointed arrows, activation; blunt arrows, inhibition.

Article Snippet: Membranes were incubated overnight at 4 °C with mouse monoclonal phospho-ERK antibody (1:1000 in 5% dried milk; Santa Cruz Biotechnology, Santa Cruz, CA), mouse monoclonal phospho-IκB-α antibody (1:1000 in 5% dried milk; Cell Signaling Technology Inc., Danvers, MA) or rabbit polyclonal Dok1 and Dok2 antibodies (1:500 in 5% BSA; Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Phospho-proteomics, Activation Assay, Expressing, Binding Assay, Activity Assay, Inhibition

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Anti-inflammation conferred by stimulation of CD200R1 via Dok1 pathway in rat microglia after germinal matrix hemorrhage

doi: 10.1177/0271678x17725211

Figure Lengend Snippet: Figure 5. The adverse effects of silencing endogenous CD200R1 or Dok1 by CD200R1 siRNA or Dok1 siRNA at 24 h after GMH. (a) Representative western blot bands. (b) Quantitative analysis of CD200 showed that protein level of CD200 increased following the treatment with CD200Fc. (c) Quantitative analysis of CD200R1 showed CD200R1 increased in the treatment group and decreased in GMH group. CD200R1 siRNA intervention significantly decreased CD200R1 level. However, CD200R1 level does not change with Dok1 siRNA intervention. (d) Quantitative analysis of Dok1 showed that Dok1 expression significantly decreased by both Dok1 siRNA and CD200R1 siRNA intervention. Data are expressed as mean SD, *P < 0.05 vs Sham, #P < 0.05 vs vehicle, &P < 0.05 vs Treatment, n ¼ 6/group, one-way ANOVA followed by the Tukey test.

Article Snippet: Primary antibodies used were CD200 (Santa Cruz Biotechnology), CD200R1 (Santa Cruz Biotechnology), Dok1 (Santa Cruz Biotechnology), IL-1beta (Abcam), and TNFalpha (Abcam).

Techniques: Western Blot, Expressing

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Anti-inflammation conferred by stimulation of CD200R1 via Dok1 pathway in rat microglia after germinal matrix hemorrhage

doi: 10.1177/0271678x17725211

Figure Lengend Snippet: Figure 6. Effects of CD200R1 siRNA and Dok1 siRNA on IL-1beta and TNF-alpha expression in the presence of CD200Fc (1.5 mg/ kg) at 24 h after GMH. (a) Representative western blots bands. (b) Quantitative analysis of IL-1beta. GMH enhanced while CD200Fc reduced the protein level of IL-1beta. (c) Quantitative analysis of TNF-alpha. GMH enhanced protein level of TNF-alpha while CD200Fc attenuated this induction. Data are expressed as mean SD, *P < 0.05 vs Sham, #P < 0.05 vs GMH þ Vehicle, &P < 0.05 vs Treatment, n ¼ 6/group, one-way ANOVA followed by the Tukey test.

Article Snippet: Primary antibodies used were CD200 (Santa Cruz Biotechnology), CD200R1 (Santa Cruz Biotechnology), Dok1 (Santa Cruz Biotechnology), IL-1beta (Abcam), and TNFalpha (Abcam).

Techniques: Expressing, Western Blot