Supporting our previous observations (Hao 0

Supporting our previous observations (Hao 0.001) are indicated. mice are sterile Kainic acid monohydrate without presenting other detectable defects, suggest that these kinases could be used as targets for male contraception. and on its dependence on magnesium ions when using myelin basic protein (MBP) and an MBP-derived peptide as a phosphorylation substrate. Materials and Methods Antibodies and other reagents All chemicals were obtained from Sigma (St. Louis, MO, USA) unless otherwise stated. Protein molecular weight standards and other reagents used for SDSCpolyacrylamide gel electrophoresis (PAGE) were from Bio-Rad (Hercules, CA, USA). Protease inhibitors were from Roche Applied Science (Indianapolis, IN, USA). Enhanced chemiluminescence (ECL) and ECLplus chemiluminescence detection kits were from GE Healthcare (Piscataway, NJ, USA). Polyvinylidene difluoride (PVDF) membrane was from Millipore (Bedford, MA, USA). His-tagged, human recombinant proteins rhTssk1, rhTssk2 (Invitrogen Corporation, Carlsbad, CA, USA), and glutathione-(sense: 5-AAA CTT GGG AGA GGG CTC AT-3 and antisense: 5-TGG CCA GAA TCT CAA TCT CC-3); (sense: 5-CCA CGC TCC AAG AAC CTA AC-3 and antisense: GAA GGA GGC AGA AGA CAT GG-3); (sense: 5-GAT GCT GGA GTC AGC AGA TG-3 and antisense: 5-GGC AAT AGC GAA TAG CCT CA-3); Universal (sense: 5-CTG TCA AGA TCA TCT CGA AG-3 and antisense: 5-GAG CCA CGT CCA AAA TGA TGT-3); (sense: 5-CGC TCA AGA TCA CGG ATT TC-3 and antisense: 5-AGG CTC CAC ACG TCG TAT TT-3); (sense: 5-GAC GAT GCT CCC CGG GCT GTA TTC-3 and antisense: 5-TCT CTT GCT CTG GGC CTC GTC ACC) and (sense: 5-TGA AGC AGG CAT CTG AGG G-3 and antisense: 5-CGA AGG TGG AAG AGT GGG AG-3). First, primers were tested by regular RTCPCR to determine specificity for each Tssk, and by using plasmid constructs carrying each Tssk open reading frame (ORF; positive control) or the corresponding vacant vector (unfavorable control). Each real-time PCR primer set was designed so that the amplimer size ranged between 150 and 250 bp. Total murine tissue RNA was obtained either from Applied Biosystems/Ambion (liver, brain, thymus, heart, lung, spleen, testicle, ovary, kidney and embryo) or from OriGene (muscle). cDNA was synthesized from RNA of murine tissues using the SuperScript III Kainic acid monohydrate kit (Invitrogen Corporation) and oligo(dT)20 according to the manufacturers’ instructions, and cDNA tested with the (Takara Bio Inc.) plus 10 M of the corresponding set of sense and antisense primer for each Tssk. Reactions were run on a Stratagene M 3000P? instrument using the following parameters: 95 for 10 s; 95 for 5 s and 60 for 22 s for 40 cycles; 95 for 1 min, 55 for 30 s and 95 for 30 s. For each assay, duplicate reactions were run and or or normalizing gene). The method was then applied for data analysis using the Stratagene M 3000P QPCR Software?, where values of each Tssk expressed in each tissue were compared with Kainic acid monohydrate those of housekeeping genes or in the testis were used as the calibrator (Livak and Schmittgen, 2001). A similar procedure was used for analyses of during testis development, using mouse testis total RNA isolated from mice of 7, 14, 21, 24 and 28 days of age. All real-time PCR data are expressed as the means SEM using Kainic acid monohydrate testis (calibrator) values as 1 arbitrary unit = 100%. Molecular cloning of mouse and “type”:”entrez-nucleotide”,”attrs”:”text”:”NM_032004″,”term_id”:”953768317″,”term_text”:”NM_032004″NM_032004 for and were amplified by PCR using the following Rabbit Polyclonal to SHP-1 (phospho-Tyr564) oligonucleotides: (sense 5-CAG CCT CGG AGG CAC TGG-3 and antisense 5-CCC TGG GCT GCA AGA GGC-3); (sense 5-CCC ACA GTG GGA ATG AGG-3 and antisense 5-CTG CAC.