Pamm 150z

To identify the differentially regulated cytokines and chemokines by PTPN2, the control or PTPN2-knockdowned cells were incubated for 4 hr with LPS (1 μg/ml). In addition, total RNA was extracted with TRIzol reagent (Invitrogen, Carlsbad, CA) and analyzed with mouse RT² profiler PCR array in accordance with the manufacturer’s protocol (PAMM-150Z, Qiagen, Valencia, CA). To determine the cytokine levels in cultured RAW264.7 cell, supernatants were collected at 6 hr (TNF-α) or 24 hr (IL-6 and IL-1β) after LPS stimulation. ATP (3 mM) was cotreated with LPS to induce the secretion of IL-1β into the culture media. The ELISA kits for measurement of mouse IL-6, IL-1β and TNF-α were supplied from R&D Systems (Minneapolis, MN). Assays were carried out in accordance with the manufacturer’s instruction. The absorbance at 450 nm was measured using a Microplate reader (BioTek Instrument, Winooski, VT). Experiments were performed in triplicates to obtain statistical significance.

Mouse cytokine and chemokine PCR array (Qiagen, Cat# PAMM-150Z, http://www.sabiosciences.com/rt_pcr_product/HTML/PAMM-150A.html) was utilized to evaluate the expression of genes encoding major pro- and anti-inflammatory cytokines and chemokines in the RNA samples. At 7 days after dMCAO, three brains per experimental group (inhibitor and control) were used to generate separate sample triplicates for the analyses. Brain cortices were dissected and stored in RNAlater solution (Ambion). Total RNA was isolated using mirVana miRNA (AB/Ambion) isolation kit, from the hemispheres ipsilateral to dMCAO injury. All measurements and data quantification were performed by Qiagen Company Sample & Assay Technologies team. The obtained raw data were analyzed using RT2 Profiler software (Qiagen). Only the genes with consistent expression levels (within the triplicate samples) were picked up for statistical analysis. The fold changes of gene expressions (inhibitor vs control) were calculated, and the transcripts that showed ≥2-fold change in expression (either up- or downregulated) were retained. At the final step, statistical significance (p value <0.05) and reliability of the results was automatically evaluated. The raw data are deposited in the Open Science Framework general data repository, link: https://osf.io/3zhc4/?view_only=0826f6e687884b90ab774328c2746ae1.