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. 2018 Sep 28;37(24):e99763. doi: 10.15252/embj.201899763

Figure EV2. Validation of phosphospecific antibodies.

Figure EV2

  • A–C
    HEK293 cells co‐transfected with full‐length WT or KD FLAG‐CDKL5 and Strep‐ARHGEF2 (A), HA‐EB2 (B) or HA‐MAP1S (C) are probed with their respective phosphospecific antibodies. Phosphospecific antibodies do not detect phosphomutants, indicating their specificity. ARHGEF2 pS122 (A) and MAP1S pS786 (C) are increased when WT CDKL5 is expressed. High levels of endogenous EB2 pS222 (B) and MAP1S pS812 (C) are not altered with WT CDKL5. Total levels of kinase (FLAG) and substrate (Strep/HA) are detected by epitope tags.
  • D, E
    Full molecular weight range of Western blots with mouse P20 cortical lysates probed for total EB2 and EB2 pS222 (D) and MAP1S light chain and MAP1S pS812 (E).
  • F
    Efficient shRNA‐mediated knockdown of EB2 in rat primary neurons is shown by the specific loss of EB2 staining in transfected cells. Scrambled shRNA was used as a control. EB2 pS222 signal is apparent in dendrites of the control (arrowheads). The remaining nuclear signal of EB2 pS222 after shRNA‐mediated knockdown (*) is considered non‐specific. Scale bar is 10 μm.

Source data are available online for this figure.