Cell line nucleofector kit t

The knock-in vectors were transfected by GenePulser (Bio-Rad Laboratories) as previously described.²⁴ Vectors were linearized by NotI, except that SalI and ScaI were used for HC constant region knock-in and the hV_H knock-in vector, respectively. RMCE donor vectors were transfected using Nucleofector 2b (Lonza) and Cell Line Nucleofector Kit T (Lonza). A total of 1 × 10⁷ cells were collected and transfected with 8 μg of DNA mixture (7 μg of RMCE construct and 1 μg of Cre recombinase expression vector) with the Nucleofector 2b (Lonza) optimized transfection program B-023.

For pull-down studies, A549 cells were transfected with individual constructs using a Nucleofector 2b device (Lonza) and Cell Line Nucleofector kit T (Lonza). Two days posttransfection, the cells were lyzed by radioimmunoprecipitation assay (RIPA) buffer (Sigma) supplemented with a protease inhibitor cocktail (Sigma) for 20 min at 4°C. Lysates were precleared by centrifuge at 1000g for 10 min, and 300 μg of the precleared lysates were incubated with 4 μg of antibodies and 50 μl of a protein A/G-tagged microbead premix (MACS) at 4°C for 1 h. The mixtures were then loaded on μ Columns (MACS) holding a μMACS Separator to retain the microbeads on the columns. The beads were washed four times with 4°C 50% RIPA buffer. Finally, bead-binding proteins were eluted by a preheated 95°C LDS sample buffer.

NALM6_GL cells were transfected with the Cell Line Nucleofector Kit T (catalog VCA-1002), Program T-001, Lonza Bioscience), and 2 μg pcDNA3.1-GPC2 or pcDNA3.1-CD276. Transfected cells were then selected by G418 or hygromycin for 7 days individually. The resultant bulk cell populations were separately stained with an anti-GPC2 antibody (clone CT3) or an anti-CD276 antibody (Abcam, clone EPNCIR122), and then sorted into high-expressing cell lines using the FACSAria (BD Biosciences). The bulk cell populations were then single-cell cloned on 96-well plates to create clones with GPC2 or CD276 expression.

Rabbit monoclonal antibodies against Phospho-STAT1 (Tyr701), Stat1, GAPDH, and Phospho-STAT1 (Tyr701) antibody (phycoerythrin conjugate) were purchased from Cell Signaling Technology. Rabbit monoclonal antibody against TC-PTP (ab129070) was purchased from Abcam. Polyethylenimine was ordered from Sigma. EGF receptor peptide (DADE-pY-LIPQQG) was purchased from GenScript. Cell Line Nucleofector Kit T was from Lonza. eBioscience Fixable viability Dye eFluor 780 (Invitrogen) and IC fixation buffer were purchased from Thermo Fisher Scientific.

NCI-H358 cells were obtained from American Type Culture Collection and maintained in RPMI1640 (Corning) supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin at 37 °C in 5% CO₂ atmosphere. For inhibitor treatment, the cells were pretreated with 30 μM compound #182 for 30 min and then stimulated with or without 1000 U/ml hIFN-γ for the indicated time. For transfection of NCI-H358 cells with pCG-TC-PTP vectors, five million cells were transfected with 3 μg of DNA using Cell Line Nucleofector Kit T (Lonza), program X-001. At 24 h, the cells were starved for another 12 h and then stimulated with or without IFN-γ for the indicated time.

For transient transfection, U251-MG cells were cultured, harvested and 1 × 10⁶ cells per cell line were co-transfected with TK(NF-κB)₆LUC vector (1 µg,^{56 (link)}) and pRLcmv vector (2 µg, Promega Corporation), using the Nucleofector II device (Lonza Group, Basel, Switzerland) via program T-027 and Cell Line Nucleofector Kit T (Lonza Group), according to manufacturer’s guidelines. PmaxGFP (Lonza Group) served as transfection control. After transient transfection, cells were firstly cultivated for 48 h in growth medium and afterwards treated for further 24 h with stimulation medium, containing TNFα (final concentration 10 ng/ml, Merck). Subsequently, analysis of NF-κB activation in U251-MG cells were performed via assessment of luciferase activities, by using the Dual-Luciferase Reporter Assay System Kit and the GloMax-Multi + Detection System, according to the guidelines of Promega Corporation. All statistical analysis were performed via Prism V5.01 software (GraphPad Software). Ratios of the dual luciferase reporter assay were determined via normalisation of the firefly luciferase activity to the corresponding amount of the Renilla activity. Subsequently, fold changes for the different cell lines and conditions were examined in relation to the untreated U251-MG PLEKHG5 wildtype.