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. Author manuscript; available in PMC: 2014 Jul 29.
Published in final edited form as: Adv Exp Med Biol. 2012;763:149–170. doi: 10.1007/978-1-4614-4711-5_7

Figure 3.

Figure 3

Components of the Scribble protein complex in the testis. Scribble, Lgl2 and Dlg1 were detected in the testis, as well as in Sertoli (isolated from 20-day-old rat testes after 4 days in culture with negligible germ cell contamination) and germ cells (isolated from 90-day-old rats after ∼12-hr in culture) by RT-PCR. Primers used in PCR were as follows: (i) Scribble: sense, 5′-CTGGCACTGCTCACAGATCT-3 (nucleotides 724-743); antisense, 5′-AGCACCTCAAGATGATTCCG (nucleotides 961-980) (GenBank Accession #:XM_002726943.1); (ii) Lgl2: sense, 5′-TCCACCATCTCGAACACTCG (nucleotides 442-461); antisense, 5′-TGCTGGATGACAACAGCCTG (nucleotides 726-745) (GenBank Accession #:NM_001127549.1); (iii) Dlg1: sense, 5′-GTTGACCTCAGAGCTGCAAG (nucleotides 2000-2019); antisense, 5′-CCACCACTCGTCATCAGAAG (nucleotides 2301-2320) (GenBank Accession #:NM_012788.1); which were co-amplified with (iv) S16: sense, 5′-TCCGCTGCAGTCCGTTCAAGTCTT (nucleotides 177-200); antisense, 5′-GCCAAACTTCTTGGATTCGCAGCG (nucleotides 538-561) (GenBank Accession #:XM_001078234). RT-PCR was performed essentially as earlier described.138,139 The cycling parameters for PCR reaction were: denaturation at 94 °C for 1 min, annealing at 57 °C for 2 min, and extension at 72 °C for 3 min, for a total of 21-25 cycles, which were followed by an extension period of 15 min at 72 °C. The identity of the PCR product was confirmed by direct nucleotide sequencing at Genewiz. SC, Sertoli cells; GC, germ cells, bp, base-pair.