cDNA synthesis and PCR were performed as described before.30 (link) The following primer sets were ordered from Eurogentec (Maastricht, The Netherlands): PRKCZ foreward 5′-GGGGGACATCTTCATCA-3′, reverse 5′-CTCGGGAAAACATGAATG-3′ PRKMZ (encoding PKMζ) forward 5′-GGCCTTCCGTTAAATA-3′, reverse 5′-ATTCGGCTTTCTTCTCCT-3′ and Ribosomal Protein S20 (RPS20) forward 5′-AAGGGCTGAGGATTTTTG-3′, reverse 5′-CGTTGCGGCTTGTTAG-3′. RPS20 expression was used as a control for cDNA input.
PCR was run at the following temperatures: 2 min 50 °C, 10 min 95 °C, 40 × (15 s 95 °C and 1 min 60 °C). PCR products with 0.1% Orange G (Merck) and 3% Ficoll (Pharmacia, Stockholm, Sweden) were run on a 1% agarose (Roche Applied Science, Almere, The Netherlands) gel containing 1:30 000 GelRed (Biotium, Hayward, CA, USA). Gels were scanned with a Gel Doc XR imager and Quantity One v4.6.3 software (Bio-Rad Laboratories, Hercules, CA, USA).
PCR was run at the following temperatures: 2 min 50 °C, 10 min 95 °C, 40 × (15 s 95 °C and 1 min 60 °C). PCR products with 0.1% Orange G (Merck) and 3% Ficoll (Pharmacia, Stockholm, Sweden) were run on a 1% agarose (Roche Applied Science, Almere, The Netherlands) gel containing 1:30 000 GelRed (Biotium, Hayward, CA, USA). Gels were scanned with a Gel Doc XR imager and Quantity One v4.6.3 software (Bio-Rad Laboratories, Hercules, CA, USA).
