Journal: Proteomes

Article Title: Evaluation of the Phosphoproteome of Mouse Alpha 4/Beta 2-Containing Nicotinic Acetylcholine Receptors In Vitro and In Vivo

doi: 10.3390/proteomes6040042

Figure Lengend Snippet: In vitro phosphorylation of α4/β2 nAChRs by CaMKII or PKA. The α4 and β2 nAChR subunits were co-expressed in HEK cells, isolated by immunoprecipitation, and subjected to mass spectrometry. Phosphorylation level was normalized to total subunit protein. ( a ) At baseline, there was a high level of phosphorylation of S470, S530, and S540 on the α4 subunit, and incubation with lambda phosphatase dephosphorylated S540 and S543 to undetectable levels. ( b ) Incubation with CaMKIIα in the presence of calcium and calmodulin increased phosphorylation of T417 and S468 on the α4 subunit significantly. ( c ) Incubation with PKA in the presence of cyclic AMP increased phosphorylation of S470, S491, and S521 significantly. * p < 0.05; *** p < 0.005. Error bars represent standard error of the mean; n = 6/condition.

Article Snippet: The remaining three groups were harvested in the absence of phosphatase inhibitors, and immunoprecipitated receptors were subject to in vitro dephosphorylation with purified lambda phosphatase (New England Biolabs, Ipswich, MA, USA; all but baseline group received this treatment).

Techniques: In Vitro, Isolation, Immunoprecipitation, Mass Spectrometry, Incubation

Journal: Molecular Biology of the Cell

Article Title: Co-regulation of the antagonistic RepoMan:Aurora-B pair in proliferating cells

doi: 10.1091/mbc.e19-12-0698

Figure Lengend Snippet: FIGURE 5: The SCFFBXW7 complex promotes RepoMan degradation in interphase. (A) EGFP traps of lysates from nonsynchronized HEK293T cells coexpressing 3xFlag-FBXW7α and either EGFP-tagged β-gal or EGFP-RepoMan-S893D were processed for immunoblotting. (B) In vitro ubiquitination of His-RepoMan by recombinant SCFFBXW7. The reaction was performed in the presence of E1 (UBE1) and E2 (UBCH3) at 30°C for 90 min. RepoMan-ubiquitination was detected by immunoblotting (IB) for both RepoMan and ubiquitin, as indicated. (C) Immunoblot analysis of lysates from U2OS cells arrested in G1/S phase nontransfected or transfected with 3xFlag-FBXW7α and siCTR (-) or 3xFlag-FBXW7α and siFBXW7 (+). *Residual band after reprobe of the blot for GAPDH. (D) Immunoblot analysis of EGFP traps from nonsynchronized HEK293T cells expressing EGFP-RepoMan-S893D and one of the indicated mutants of FBXW7α. (E) EGFP-RepoMan-S893D traps from nonsynchronized HEK293T cells were preincubated with buffer or lambda phosphatase (lambda PP) and examined for retained ectopically expressed FBXW7α. (F) Schematic representation of the predicted and established CDK phosphorylation sites of human RepoMan. Red, established phosphorylation site (Dephoure et al., 2008; Olsen et al., 2010; Vagnarelli et al., 2011; Prévost et al., 2013; Qian et al., 2015). Black, CDK phosphorylation sites, as determined by mass spectrometry (Wu et al., 2018); gray, CDK phospho-sites predicted by NetPhos 3.1 (Blom et al., 2004). (G) HEK293T cells that transiently expressed EGFP-RepoMan-S893D and 3xFlag-FBXW7α were arrested in G1/S with a single thymidine block and treated for 4 h with DMSO or 20 μM roscovitine to examine the effect on the retention of FBXW7α by EGFP-traps. (H) Effect of the deletion of residues 400–550 (∆400–550) on the binding of ectopically expressed 3x-Flag-FBXW7α and endogenous CDK2 to EGFP-RepoMan-S893D in G1/S HEK293T cells. (I) Immunoblot analysis of lysates from G1/S HEK293T cells expressing EGFP-tagged RepoMan-S893D or ∆400-550 in the absence (-) or presence (+) of 3x-Flag-FBXW7α. (J) Relative abundance of EGFP-RepoMan levels from five independent experiments normalized to GAPDH and to the control (no transfection of 3xFlag-FBXW7α). Ns, not significant; **P value < 0.01 in paired t test. (K) Cartoon of the SCFFBXW7 complex associated with phosphorylated RepoMan (RM). Adapted from Crusio et al. (2010). SKP1, S-phase kinase-associated protein 1; CUL1, Cullin-1; RBX1, E3 ubiquitin-protein ligase RBX1.

Article Snippet: For the lambda phosphatase treatment, EGFP traps were treated with λ phosphatase (Santa Cruz) for 30 min at 30°C.

Techniques: Western Blot, In Vitro, Ubiquitin Proteomics, Recombinant, Transfection, Expressing, Phospho-proteomics, Mass Spectrometry, Blocking Assay, Binding Assay, Control

Journal: The Journal of Neuroscience

Article Title: Trip6 Promotes Dendritic Morphogenesis through Dephosphorylated GRIP1-Dependent Myosin VI and F-Actin Organization

doi: 10.1523/JNEUROSCI.2125-14.2015

Figure Lengend Snippet: GRIP1956T is phosphorylated by AKT1. A, GFP-LR2+PDZ7 (GRIP1 fragment) was immunoprecipitated (IP) with anti-GFP antibody and separated by SDS-PAGE followed by mass spectrometry analysis. Threonine at the 956 position (956T) was identified to be phosphorylated (red box). Co. St., Coomassie blue staining. B, Alignment of the amino acid sequence containing GRIP1855T (negative control) and GRIP1956T among different eukaryotes; 956T is emphasized in bold and marked by an asterisk; the predicted AKT1-phosphorylated motif is indicated by the arrow. C, Overexpressed GFP-LR2+PDZ7 (GRIP1 fragment) was immunoprecipitated from HEK293T cells with anti-GFP antibody and treated with lambda phosphatase (λ PPase) or calf intestine alkaline phosphatase (CIAP) followed by immunoblotting (IB) with anti-GRIP1956T phosphorylation-specific antibody (pGRIP1). D, WT and phosphodead mutants T855A, T956A, and both T855A and T956A (T855A+T956A) of GFP-LR2+PDZ7 were transfected into HEK293T cells and analyzed by IB with pGRIP1 antibody. E, F, IB of phospho-GRIP1956T (pGRIP1) and total GRIP1 (tGRIP1) in the whole-brain (E) and hippocampus (F) homogenates of mice at different developmental stages. E, Embryonic day; P, Postnatal day; PW, Postnatal week. G, Pull-down of overexpressed GFP-AKT1 by GST-LR2+PDZ7 in HEK293T cell lysates. H, GFP-LR2+PDZ, together with AKT1 shRNA vector (628 or 642), was transfected into HEK293T cells for 72 h followed by IB. I, J, HEK293T cells overexpressing WT (I) or T956A (J) of GFP-LR2+PDZ7 were treated with insulin alone or together with wortmannin following serum starvation for 36 h and then analyzed by IB with pGRIP1 antibody. K, Cultured hippocampal neurons were treated with insulin alone or together with wortmannin following serum starvation at 7 DIV for 36 h and then analyzed by IB with pGRIP1 antibody.

Article Snippet: One aliquot was left untreated, and the other two were washed extensively with their respective buffers before treatment either with 400 U lambda phosphatase (Merck) at 30°C for 30 min or with 400 U of calf intestinal alkaline phosphatase (Promega) at 37°C for 30 min.

Techniques: Immunoprecipitation, SDS Page, Mass Spectrometry, Staining, Sequencing, Negative Control, Western Blot, Transfection, shRNA, Plasmid Preparation, Cell Culture

Journal: PLoS ONE

Article Title: Genetic Disruption of the Sh3pxd2a Gene Reveals an Essential Role in Mouse Development and the Existence of a Novel Isoform of Tks5

doi: 10.1371/journal.pone.0107674

Figure Lengend Snippet: (A) Tks5 immunoblotting of extracts from Src-transformed NIH3T3 cells and their control counterparts expressing empty vector or Tks5β. Number on the left indicate kDa. Asterisks indicate Tks5 middle (140 kDa) and low (130 kDa) bands. (B) Tks5 immunoblotting on extracts from Src-transformed NIH3T3 cells expressing an inducible Tks5 short isoform with or without induction with Doxycycline for 24 h. Number on the left indicates kDa. Asterisks indicate Tks5 middle (140 kDa). (C) Tks5 immunoblotting on extracts from Src-transformed NIH3T3 cells expressing empty vector or Tks5β upon treatment with a 5 µM concentration of the indicated Src family kinase inhibitors for 16h. Number on the left indicates kDa. Asterisks indicate Tks5 middle and low bands corresponding to non-PX containing Tks5 isoforms. (D) Tks5 immunoblotting on extracts from Src-transformed NIH3T3 cells untreated or expressing empty vector or Tks5β upon treatment with lambda protein phosphatase in the presence or absence of sodium orthovanadate. Asterisk indicate Tks5 middle (140 kDa) band.

Article Snippet: Cell lysates were immunoprecipitated for Tks5 using 6G1 antibody and lambda protein phosphatase treatment (Cell Signaling Technologies) was performed after the last immunoprecipitation wash. Phosphatase was added according with the manufacturer's instructions in the presence or absence of 100 μM of sodium orthovanadate.

Techniques: Western Blot, Transformation Assay, Control, Expressing, Plasmid Preparation, Concentration Assay