Mini spin kit

Total RNA was extracted from cultured cells using Mini spin kit (Macherey-Nagel). RNA extracted from canine fibroblasts was obtained from Prof. Hannes Lohi group. 1000 ng of RNA was reverse transcribed using Maxima First Strand cDNA Synthesis Kit for RT-qPCR (Thermo Fisher scientific). Real-time PCR analysis was done by DyNAmo Flash SYBR Green qPCR Kit (Thermo Fisher scientific) using CFX96 TM Real-Time PCR Detection System (Bio-Rad). The PCR program started with 95°C for 7 min followed by 40 cycles at 95°C for 10 s and 60°C for 30 s. Beta-2-Microglobulin (B2M) was used as a reference gene for normalization and mRNA expression level was calculated using the comparative Ct (threshold cycle) method. All primer sequences are in the Table 1.

To clone the NERCLIN cDNA we extracted total RNA from 143B cells using Mini spin kit (Macherey-Nagel). Then RNA was reverse transcribed using Maxima First Strand cDNA Synthesis Kit for RT-qPCR (Thermo Fisher scientific). The cDNA was PCR amplified using specific primers for TOPO cloning. Following gel electrophoresis the correct band was cut and the DNA was extracted from the gel for TOPO cloning. The TOPO construct containing NERCLIN was used as a template for cloning NERCLIN into EGFP or pBabe vectors.
To fuse BirA* to C-terminus of NERCLIN we used PCR overlap extension. pHA-BirA* and pBabe-NERCLIN constructs were used the templates. The resulted fused construct was inserted into pBabe vector using EcoRI and SalI restriction sites. GFP-BirA* and AIF-BirA* plasmids were as in 23 .