Total RNA from Con (n = 5) samples was isolated with TRNzol-A+ reagent (TIANGEN, Beijing, China) according to the manufacturer’s instructions. cDNA was synthesized with DNAse I (Fermentas) treated total RNA using the BioRT cDNA First Stand Synthesis Kit (Bioer Technology, Hangzhou, China). Primers used for RT-PCR are listed in Table S1 . Q-PCR was performed using the BioEasy SYBR Green I Real Time PCR Kit (Bioer Technology, Hangzhou, China) with the BIO-RAD Iq5 Multicolor Real-Time PCR Detection System. The relative gene expression normalized to the GAPDH was determined by 2−ΔΔCT formula. All the data of gene expression were performed three times.
For Western blotting, the eyes tissues from BR, WR and 3′ UTR KO rabbits were homogenized in 150 lL of lysis buffer. The protein concentrations were measured by the Braford method (Bio- Rad). In this study anti-TYR polyclonal antibody (1:2000; abcam) and anti-GAPDH monoclonal antibody (1:2000; Beyotime) were used as primary and internal control respectively. Images were quantified using ImageJ software (NIH) and all data are expressed as mean ± SEM.
For Western blotting, the eyes tissues from BR, WR and 3′ UTR KO rabbits were homogenized in 150 lL of lysis buffer. The protein concentrations were measured by the Braford method (Bio- Rad). In this study anti-TYR polyclonal antibody (1:2000; abcam) and anti-GAPDH monoclonal antibody (1:2000; Beyotime) were used as primary and internal control respectively. Images were quantified using ImageJ software (NIH) and all data are expressed as mean ± SEM.