Silencing BNP and Cdk5 in Trigeminal Neurons

Cultured mouse trigeminal neurons were transfected with specific siRNA BNP (siBNP) oligonucleotides (Sigma), or siCdk5 oligonucleotide (ThermoFisher Scientific, Milan, Italy) using the DharmaFECT transfection reagent (Dharmacon, Lafayette, CO, USA). As control (“scramble”), cells were transfected with siGLO RISC-Free siRNA (Dharmacon). The BNP siRNA and control siRNA were transfected at a final concentration of 100 nM for 24 h in triplicate for each treatment. At 48 h post-transfection, BNP knockdown was confirmed by immunostaining and Elisa assay. A pool of two different oligonucleotide sequences were used to knockdown BNP (NM_008726) expression, as previously published [76 (link)]: BNP siRNA-1: sense 5′-CCCAGAGACAGCUCUUGAATT-3′ with antisense 5′-UUCAAGAGCUGUCUCUGGGTT-3′, and BNP siRNA-2: sense 5′-GGCACAAGAUAGACCGGAUTT-3′ with antisense 5′-AUCCGGUCUAUCUUGUGCCTT-3′. To knockdown Cdk5 expression (NM_007668.3), siCdk5 or control siRNA sequences were transfected at a final concentration of 100 nM. Twenty-four hours after silencing, cells were used for protein expression and patch clamping experiments. Efficiency of Cdk5 silencing was tested with Western blot.