Journal: The EMBO Journal

Article Title: Chemical genetic identification of CDKL 5 substrates reveals its role in neuronal microtubule dynamics

doi: 10.15252/embj.201899763

Figure Lengend Snippet: 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.

Article Snippet: Rabbit polyclonal phosphospecific antibodies were raised against the following phosphorylated (*) peptides by Covalab: TTRERPTS*AIY (mouse ARHGEF2 pS122), PGSTPSRPSS*AKRA (mouse EB2 pS222), RKAPARPSS*ASAT (mouse MAP1S pS786), AGDRNRPLS*ARSE (mouse MAP1S pS812).

Techniques: Transfection, Molecular Weight, Western Blot, shRNA, Knockdown, Staining, Control

Journal: The EMBO Journal

Article Title: Chemical genetic identification of CDKL 5 substrates reveals its role in neuronal microtubule dynamics

doi: 10.15252/embj.201899763

Figure Lengend Snippet: A, B In vivo substrate phosphorylations are assessed by Western blotting of 5 WT (CDKL5 + /Y) and 5 CDKL5 KO (CDKL5 − /Y) mouse cortical lysates using phosphospecific antibodies. EB2 pS222 and MAP1S pS812 are dramatically reduced in KOs. Quantification of substrate phosphorylation is normalized for total protein level. Student's t ‐test: n = 5 animals per genotype. C, D Developmental expression of CDKL5 and levels of its substrate phosphorylations in P4–P50 cortex. CDKL5‐dependent substrate phosphorylation is quantified by subtracting KO from WT levels for each age. n = 3 animals per age per genotype. E, F Expression of CDKL5 and levels of substrate phosphorylation in P8 Nex‐Cre conditional KO cortex. CDKL5 protein expression is almost completely lost. Quantification of substrate phosphorylation is normalized for total protein level. Student's t ‐test: n = 3 animals per genotype. G CDKL5 WT and CDKL5 KO neurons in culture are co‐stained with neuronal dendrite marker MAP2, EB2 and EB2 pS222. EB2 pS222 is lost in CDKL5 KO dendrites. Scale bar is 20 μm. Data information: EB2 isoform 1/2 (37 kD) is quantified. **** P < 0.0001, error bars are SEM. Source data are available online for this figure.

Article Snippet: Rabbit polyclonal phosphospecific antibodies were raised against the following phosphorylated (*) peptides by Covalab: TTRERPTS*AIY (mouse ARHGEF2 pS122), PGSTPSRPSS*AKRA (mouse EB2 pS222), RKAPARPSS*ASAT (mouse MAP1S pS786), AGDRNRPLS*ARSE (mouse MAP1S pS812).

Techniques: In Vivo, Western Blot, Phospho-proteomics, Expressing, Staining, Marker

Journal: Molecular cell

Article Title: Arc oligomerization is regulated by CaMKII phosphorylation of GAG domain; an essential mechanism for plasticity and memory formation.

doi: 10.1016/j.molcel.2019.05.004

Figure Lengend Snippet: (A) Arc immunoprecipitation from naïve adult mouse brain and MS analysis. (B) Immunoprecipitates from detergent lysates of WT and Arc KO brain were sequentially eluted with 2% SDS and 2% SDS with 10 mM BME and analyzed by Coomassie. Arc band was excised and submitted for MS. Yellow arrows: Arc, Red arrows: Arc knockout control. (C) Mass spectrometry profile of the sequence of Arc from residues 257-268 is shown. Analysis by MS/MS revealed the presence of phosphorylated modification. The fragment ions whose m / z value corresponds to y or b ions (peptide fragments from C-terminus and from N-terminus, respectively) are indicated. The observed fragment ions are labeled on the spectrum and peptide sequence. (D) schematic of Arc indicating position of phosphorylation sites. (E) CaMKII phosphorylates ArcS260. Arc or ArcS260A were co-transfected with constitutively active CamKIIα (CamKII T286D) to HEK293 cells and cell lysates were blotted with ArcS260 phospho-specific antibody. (F) ArcS260 is phosphorylated in mouse brain. A parallel blot with Arc monoclonal antibody confirmed Arc immunoprecipitation and insensitivity to lambda phosphatase. (G) ArcS260 phosphorylation is induced in cultured neurons. Phospho-specific antibody revealed bicuculline-induced phosphorylation of ArcS260 that was blocked by 30 min pretreatment with CamKII inhibitor (KN93) but not control (KN92).

Article Snippet: Rabbit polyclonal phosphospecific antibodies for ArcS260 and ArcT278 were generated and affinity purified by AnaSpec Inc. using Arc pS260-EFKQG{pSER}VKNW, Arc pT278-QYSEG{pTHR}LSRE. .

Techniques: Immunoprecipitation, Knock-Out, Control, Mass Spectrometry, Sequencing, Tandem Mass Spectroscopy, Modification, Labeling, Phospho-proteomics, Transfection, Cell Culture

Journal: Molecular cell

Article Title: Arc oligomerization is regulated by CaMKII phosphorylation of GAG domain; an essential mechanism for plasticity and memory formation.

doi: 10.1016/j.molcel.2019.05.004

Figure Lengend Snippet: Whole-cell voltage-clamp recordings from mouse Purkinje cells expressing Arc transgenes in culture. (A) Basal mEPSC amplitude was driven low and chemical LTD evoked by PDA was occluded in Arc-transfected Purkinje cells. This effect was not blocked by pretreatment with the CaMKII inhibitor KN-93. N= 10 cells/group. (B) Phosphomimic mutant at the site S260 and C-lobe mutant R335E did not reduce the basal amplitude of mEPSCs and did not occlude subsequent PDA-evoked chemical LTD. ArcS260A mimics WT Arc by reducing mEPSC and occluding chemical LTD. N= 10 cells/group. The scale bars for the insets on (A) and (B) are both 500 msec, 20 pA. (C) Purkinje cells in primary cerebellar culture were transfected with Arc transgene or an empty vector control plasmid and examined using a standard protocol that induces dendrite-specific LTD in Purkinje cells treated with the control plasmid (n=8; or untransfected cells, not shown). In Arc expressing cells (n=7) LTD was occluded in the paired pathway that received glutamate/depolarization conjunction as indicated by the horizontal bars (paired pathway), while LTP was induced in the control pathway. LTP was abolished by pretreatment with the CamKII inhibitor KN-93 (n=8). Scale bars = 2 sec, 50 pA. (D) LTD induced by glutamate/depolarizing pairing (indicated by horizontal bars) was occluded by both WT, ArcS260A and ArcS260,T278A. LTP of control pathway was abolished in neurons expressing de-phosphorylation mimic mutants at ArcS260A. Arc plasmid (n=7); Arc S260A plasmid (n=8); Arc S260A,T278A plasmid (n=6). Scale bars = 2 sec, 60 pA.

Article Snippet: Rabbit polyclonal phosphospecific antibodies for ArcS260 and ArcT278 were generated and affinity purified by AnaSpec Inc. using Arc pS260-EFKQG{pSER}VKNW, Arc pT278-QYSEG{pTHR}LSRE. .

Techniques: Expressing, Transfection, Mutagenesis, Plasmid Preparation, Control, De-Phosphorylation Assay