antibodies against il 36γ  (Bio-Rad)

Journal: Journal of Neuroinflammation

Article Title: Interleukin-36γ is expressed by neutrophils and can activate microglia, but has no role in experimental autoimmune encephalomyelitis

doi: 10.1186/s12974-015-0392-7

Figure Lengend Snippet: IL-36γ and IL-36R are transcriptionally upregulated in 3 EAE models. Quantification of the mRNAs encoding IL-36γ and IL-36R by qRT-PCR in different tissues from mice killed at the indicated time points after induction of EAE by either active immunization with MOG peptide, passive transfer of encephalitogenic T cells, or injection of PTX to 2D2 mice. Control non-transgenic mice were injected with PBS, PTX, or CFA only. Stars indicate significant differences from the PBS group only ( white star ) or all the corresponding control groups ( black star ), as determined by Wilcoxon tests ( P ≤ 0.038). Sample size: 11–13 (PBS), 5–13 (PTX), 4–8 (CFA), 7–8 (active EAE), 5–8 (passive EAE), 6 (2D2 with EAE), or 5 (2D2 without [ w/o ] EAE)

Article Snippet: The membrane was blocked in PBS containing 0.1 % Tween 20 and 7 % non-fat milk, and then incubated at 4 °C overnight with an antibody against IL-36γ (1:200, Santa Cruz, sc-168163) or β-actin (1:50000, Abcam, mab150), followed by 1 h at room temperature in the appropriate secondary horseradish peroxidase-conjugated antibody (Cell Signaling Technology).

Techniques: Quantitative RT-PCR, Injection, Control, Transgenic Assay

Journal: Journal of Neuroinflammation

Article Title: Interleukin-36γ is expressed by neutrophils and can activate microglia, but has no role in experimental autoimmune encephalomyelitis

doi: 10.1186/s12974-015-0392-7

Figure Lengend Snippet: IL-36γ is selectively expressed by neutrophils, while IL-36R is expressed by different leukocytes such as monocytic cells. a Quantification of IL-36γ or IL36R mRNA by qRT-PCR in different cells purified from the spinal cord or spleen of EAE mice by FACS using the following gating strategies: neutrophils, Ly6G + CD11b + CD45 + CD3 − CD19 − ; microglia, CD11b + CD45 low Ly6G − CD3 − CD19 − ; monocyte-derived cells (MDC; comprising macrophages and CD11c + dendritic cells), CD11b + CD45 + CD11c +/− Ly6G − CD3 − CD19 − ; other intraspinal leukocytes, CD45 + CD3 +/− CD19 +/− CD11b − Ly6G − CD11c − ; B cells, CD19 + CD45 + CD11b − Ly6G − CD3 − ; splenic macrophages, CD11b + CD45 + CD11c − Ly6G − CD3 − CD19 − ; other splenic leukocytes, CD45 + CD3 +/− CD11b +/− CD11c +/− Ly6G − CD19 − . Sample size: spinal cord, one pooled sample of sorted cells from four mice; spleen, five non-pooled samples from individual mice. b Western blotting showing the full-length form of IL-36γ (~22 kDa) in splenic neutrophils from a wild-type EAE mouse (IL-36γ +/+ ), but not from an IL-36γ-deficient EAE mouse (IL-36γ −/− ). Data are representative of at least four mice per group. The recombinant ( truncated ) form of IL-36γ was used as a control ( right lane ). β-actin ( lower panels ) was used to control for protein loading. Asterisks indicate non-specific bands. c Autoradiograms showing in situ hybridization signals ( arrows ) for IL-36γ or IL-36R mRNA in the spinal cord of mice killed on day 12 after active EAE induction, but not in naïve mice. Note the submeningeal distribution of the signals (representative of at least five mice). Scale bar = 500 μm. d Double labeling for IL-36γ or IL-36R mRNA ( black grains , in situ hybridization) and cell type-specific markers ( red brown , immunohistochemistry) in CNS sections from EAE mice. Arrows show examples of double-labeled cells. Scale bar = 20 μm

Article Snippet: The membrane was blocked in PBS containing 0.1 % Tween 20 and 7 % non-fat milk, and then incubated at 4 °C overnight with an antibody against IL-36γ (1:200, Santa Cruz, sc-168163) or β-actin (1:50000, Abcam, mab150), followed by 1 h at room temperature in the appropriate secondary horseradish peroxidase-conjugated antibody (Cell Signaling Technology).

Techniques: Quantitative RT-PCR, Purification, Derivative Assay, Western Blot, Recombinant, Control, In Situ Hybridization, Labeling, Immunohistochemistry

Journal: Journal of Neuroinflammation

Article Title: Interleukin-36γ is expressed by neutrophils and can activate microglia, but has no role in experimental autoimmune encephalomyelitis

doi: 10.1186/s12974-015-0392-7

Figure Lengend Snippet: IL-36γ stimulates the expression of inflammatory genes in microglial cultures. a Genes that were upregulated ≥2.5 times in BV2 microglia treated for 6 h with recombinant IL-36γ (100 ng/ml) compared to PBS, as determined using Affymetrix DNA microarrays (Student’s t test, P < 0.04). b , c Quantitative PCR analysis confirming that Csf3, IL-1β, and Cxcl2 mRNAs were upregulated by IL-36γ in BV2 cells ( b ) and primary microglia from IL-36R +/+ mice ( c ) (Wilcoxon test, P < 0.005). Note in c the absence of upregulation in primary microglia from IL-36R −/− mice, confirming the specificity of the results

Article Snippet: The membrane was blocked in PBS containing 0.1 % Tween 20 and 7 % non-fat milk, and then incubated at 4 °C overnight with an antibody against IL-36γ (1:200, Santa Cruz, sc-168163) or β-actin (1:50000, Abcam, mab150), followed by 1 h at room temperature in the appropriate secondary horseradish peroxidase-conjugated antibody (Cell Signaling Technology).

Techniques: Expressing, Recombinant, Real-time Polymerase Chain Reaction

Journal: Journal of Neuroinflammation

Article Title: Interleukin-36γ is expressed by neutrophils and can activate microglia, but has no role in experimental autoimmune encephalomyelitis

doi: 10.1186/s12974-015-0392-7

Figure Lengend Snippet: Neither IL-36γ nor IL-36R is required for EAE development. a Kaplan-Meier curves showing EAE incidence in mice expressing ( black squares ) or lacking ( white squares ) IL-36γ ( upper graph ) or IL-36R ( bottom graph ) after immunization with MOG. The graphs include all the mice tested. No significant intergenotype difference was detected (Wilcoxon tests, P ≥ 0.41). Sample size: 20 (IL-36γ +/+ ), 20 (IL-36γ −/− ), 6 (IL-36R +/+ ), or 6 (IL-36R −/− ). b EAE severity in mice expressing ( black squares ) or lacking ( white squares ) IL-36γ ( upper graph ) or IL-36R ( bottom graph ). The graphs include only mice that had developed clinical signs of EAE at the end of the study (i.e., 21 days). No significant intergenotype difference was detected (Wilcoxon tests, P ≥ 0.12). Sample size: 17 (IL-36γ +/+ ), 17 (IL-36γ −/− ), 5 (IL-36R +/+ ), or 5 (IL-36R −/− ). c Additional statistics for EAE in mice expressing (+/+) or not (−/−)IL-36γ or IL-36R. No significant intergenotype difference was detected in any of these parameters (Wilcoxon tests, P ≥ 0.34). Sample size: 20 (IL-36γ +/+ ), 20 (IL-36γ −/− ), 6 (IL-36R +/+ ), or 6 (IL-36R −/− )

Article Snippet: The membrane was blocked in PBS containing 0.1 % Tween 20 and 7 % non-fat milk, and then incubated at 4 °C overnight with an antibody against IL-36γ (1:200, Santa Cruz, sc-168163) or β-actin (1:50000, Abcam, mab150), followed by 1 h at room temperature in the appropriate secondary horseradish peroxidase-conjugated antibody (Cell Signaling Technology).

Techniques: Expressing

Journal: Journal of Neuroinflammation

Article Title: Interleukin-36γ is expressed by neutrophils and can activate microglia, but has no role in experimental autoimmune encephalomyelitis

doi: 10.1186/s12974-015-0392-7

Figure Lengend Snippet: No difference in leukocyte recruitment in the spinal cord of EAE mice expressing or not IL-36γ or IL-36R. a Gating strategy used for flow cytometric analysis. Dead cells and doublets were excluded. The data shown are from a representative wild-type EAE mouse. b , c Counts of immune cells in the spinal cord of IL-36γ −/− , IL-36R −/− , or wild-type mice killed 21 days after immunization with MOG peptide. The counts were normalized to CD45 − cells used as an internal control. When the cells were collected, the animals had clinical scores ranging from 0.5 to 3. No significant difference was found between the genotypes (Wilcoxon tests, P ≥ 0.07). Sample size: 6 ( b ) or 9 ( c ) mice per group

Article Snippet: The membrane was blocked in PBS containing 0.1 % Tween 20 and 7 % non-fat milk, and then incubated at 4 °C overnight with an antibody against IL-36γ (1:200, Santa Cruz, sc-168163) or β-actin (1:50000, Abcam, mab150), followed by 1 h at room temperature in the appropriate secondary horseradish peroxidase-conjugated antibody (Cell Signaling Technology).

Techniques: Expressing, Control