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. 2021 May 3;40(12):e106393. doi: 10.15252/embj.2020106393

Figure 1. Knockdown of Phf5a inhibits CSR.

Figure 1

  1. Representative FACS profiles of IgA switching in CH12F3‐2A cells. The control and Phf5a siRNAs are indicated at the top. The number in each FACS profile indicates the percent (%) IgA+ cells at 24 and 48 h after CIT stimulation.
  2. Top: Illustration of Phf5a with a central PHD domain. Bottom: Confirmation of Phf5a KD by Western blot.
  3. CSR complementation assay using siRNA‐resistant Phf5a with Myc and Flag (MF) epitopes tagged at the C‐terminus.
  4. Schematic of MF‐tagged siRNA‐resistant Phf5a (Phf5aR‐MF). Asterisks (*) indicate the approximate locations of mutations in the cDNA made to generate the siRNA‐resistant version. Western blot confirmed the KD efficiency of the endogenous Phf5a, and the expression of Phf5aR‐MF.
  5. Quantitative RT–PCR (qRT–PCR) of µGLT, αGLT, and AID from siControl‐ and siPhf5a‐treated samples. The values are presented as mean ± SD (n = 3).
  6. Ethidium Bromide‐stained gels showing the PCR detection of excision circular DNA (αCD) and Gapdh as a control in the genomic DNA samples indicated.