Quantitative PCR (qPCR) was utilized to measure the proportion of FoxP3 mRNA splice variants. The protocols for isolating RNA, reverse transcription, primers used, amplification cycles, hardware, and software are described in detail in our previous works [29 (link),37 (link)]. The average RNA levels of three genes (beta-actin, 18S, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)) were utilized as a reference to normalize the determined mRNA levels of the FoxP3 splice variants.
The detection of the protein splice variant with exon 2 was performed by Western blotting using exon-specific FoxP3 antibodies. Clone 150D can recognize exon 2, while clone 259D (both from BioLegend, San Diego, CA, USA) is specific to the epitope located after exon 2 which is common for all splice variants. The Western blotting protocol was previously described by us [38 (link)]. GAPDH was used as a loading control. The catalog numbers for antibodies used for Western blotting are presented inTable S1 in the Supplementary Materials .
The detection of the protein splice variant with exon 2 was performed by Western blotting using exon-specific FoxP3 antibodies. Clone 150D can recognize exon 2, while clone 259D (both from BioLegend, San Diego, CA, USA) is specific to the epitope located after exon 2 which is common for all splice variants. The Western blotting protocol was previously described by us [38 (link)]. GAPDH was used as a loading control. The catalog numbers for antibodies used for Western blotting are presented in