Clonexpresstmone step cloning kit

The strains and plasmids used in this study are listed in Table 1. The noxE (GenBank Accession No. AM406671) gene encoding the water-forming NADH oxidase from L. lactis was PCR-amplified with primers Nox-F, 5′-CTTGTGGGCCCAGGATCCATGAAAATCGTAGTTATCG-3′, and Nox-R, 5′-ACAGGAATTCACCATGGATCCTTATTTGGCATTCAAAGCTG-3′. Both primers have a BamHI site (underlined), and the homologous arms of the plasmid are indicated in italics in the primer sequences. PCR products were gel-purified and inserted into the BamHI site of pYX212 by using the ClonExpress^TMOne Step Cloning Kit (Vazyme Biotech, Co., Ltd, Nanjing, China), yielding pYX212-NOX. The plasmid was transformed into the host strain, BY4741, using G418 (400 μg/ml) to select a stably transfected clone, designated NOX (Table 1). As a control, CON, the host strain transfected with empty plasmid, was used.

Human and mouse IκBα were generated by PCR amplification from the cDNA of HT-29 cells and MEFs, respectively, and then cloned into the pMSCV vector or a lentiviral vector made in-house. The S32A/S36A double mutant or K21R/K22R double mutant IκBα were created by PCR-based site-directed mutagenesis. To construct the dimerizable MLKL, human MLKL and FKBP were amplified from cDNA of HT-29 cells. T357E/S358D MLKL and F36V FKBP were generated through PCR-based site-directed mutagenesis. The mutated MLKL, F36V FKBP, and Flag tag oligonucleotides were cloned into pMSCV vector using ClonExpress^TM One Step Cloning Kit (Vazyme, Nanjing, China). The annealed shRNA and single-guide RNA (sgRNA) oligonucleotides were cloned into the pLKO.1 vector and LentiCRISPR v2 vector, respectively. All plasmids were confirmed by sequencing.
The shRNAs used in this study were selected from the RNAi Consortium (TRC) (Broad Institute, MA, USA) and are listed in Supplementary Table 1. To silence RIPK1 in MEFs, the following sgRNAs were used: sgRIPK1 #1_Sense (S): caccgGGGTCTTTAGCACGTGCATC, sgRIPK1 #1_Antisense (AS): aaacGATGCACGTGCTAAAGACCCc; sgRIPK1 #2_S: caccgAGAAGAAGGGAACTATTCGC, sgRIPK1 #2_AS: aaacGCGAATAGTTCCCTTCTTCTc; sgRIPK1 #3_S: caccgTGTGAAAGTCACGATCAACG, sgRIPK1 #3_AS: aaacCGTTGATCGTGACTTTCACAc.

Human TEX11 cDNA (GenBank No. NM_001003811) was cloned in the pcDNA vector with MYC-tag by homologous recombination via the ClonExpress^TM one-step cloning kit (Vazyme, C112). The cDNA obtained from the testis of patients with OA were used as the template for PCR amplifications. The linearized vector was obtained by digesting the circular vector with double restriction enzyme (KpnI-HF^® and EcoRI-HF^®) digestion. All deletions and substitution mutants from the TEX11 expression constructs were generated by PCR. The PCR was performed with the PrimeSTAR system (Takara, Beijing, China) using the following conditions: 98°C for 5 min, 18 cycles of 98°C for 10 s, 60°C for 5 s and 72°C for 270 s, 7 min at 72°C and forever at 4°C. All constructs were verified by sequencing before the experiments. Then 0.1 μl of DMT Enzyme (TRANS^®GD111-01 20,000 U/ml) was added to 10 μl of the reaction, mixed well, and incubated at 37°C for 1 h. The final reaction was transformed into competent cells. Colony, miniprep, and sequence were picked to check for mutation and any PCR-introduced errors. All primers used for plasmid construction are summarized in Supplementary Table 2.