myc tag 9b11 cst  (Cell Signaling Technology Inc)

Journal: eLife

Article Title: Native KCC2 interactome reveals PACSIN1 as a critical regulator of synaptic inhibition

doi: 10.7554/eLife.28270

Figure Lengend Snippet: ( a ) Schematic of the locations of anti-KCC2 antibodies. ( b ) The primary KCC2 amino acid sequence. Red indicates the protein coverage of KCC2 identified by MS analysis; yellow indicates unique coverage for KCC2a and KCC2b isoforms. MS/MS- spectra of peptides unique for KCC2a and KCC2b. Right: the MS/MS ion fragmentation of the corresponding amino acid sequence is indicated above the spectra. ( c ) Spectral and peptide count plots of proteins in AP with all three anti-KCC2 antibodies in developing brain membrane fractions (P5, left) and adult brain membrane fractions (P50, right). Peptide and spectral counts are normalized (anti-KCC2/IgG) and plotted on a log scale. Red circles - highly enriched KCC2 bait. Blue circles - highly enriched PACSIN1 target peptides. Dark-grey circles - top proteins enriched with KCC2-AP in comparison to IgG control-AP. Light-grey circles - proteins enriched in IgG control-AP in comparison to KCC2-AP and known spurious interactors. 10.7554/eLife.28270.009 Figure 2—source data 1. LC/MS replicates.

Article Snippet: Cell lysates or solubilized membrane fractions (∼0.2–0.5 mg protein) were incubated with N-terminal KCC2b or anti-myc (CST #9B11, RRID AB_331783 ) antibodies on a rotating platform (4 hr, 4°C).

Techniques: Sequencing, Tandem Mass Spectroscopy, Membrane, Comparison, Control, Liquid Chromatography with Mass Spectroscopy

Journal: eLife

Article Title: Native KCC2 interactome reveals PACSIN1 as a critical regulator of synaptic inhibition

doi: 10.7554/eLife.28270

Figure Lengend Snippet: Native KCC2 complexes from C 12 E 9 -solubilized whole-brain membrane fractions immunoprecipitated with pre-immune sera or anti-N-terminal KCC2b antibody ( a ) or anti-p Ser 940 KCC2 antibody ( b ) and immunoblotted with the antibodies indicated at right (C-terminal KCC2 antibody). Representative example of five biological replicates. IP, immunoprecipitate; I, input fraction (1% of IP); U. unbound fraction (1%of IP); O. oligomer; M. monomer.

Article Snippet: Cell lysates or solubilized membrane fractions (∼0.2–0.5 mg protein) were incubated with N-terminal KCC2b or anti-myc (CST #9B11, RRID AB_331783 ) antibodies on a rotating platform (4 hr, 4°C).

Techniques: Membrane, Immunoprecipitation

Journal: eLife

Article Title: Native KCC2 interactome reveals PACSIN1 as a critical regulator of synaptic inhibition

doi: 10.7554/eLife.28270

Figure Lengend Snippet: KCC2 protein partners identified by ME-APs

Article Snippet: Cell lysates or solubilized membrane fractions (∼0.2–0.5 mg protein) were incubated with N-terminal KCC2b or anti-myc (CST #9B11, RRID AB_331783 ) antibodies on a rotating platform (4 hr, 4°C).

Techniques:

Journal: eLife

Article Title: Native KCC2 interactome reveals PACSIN1 as a critical regulator of synaptic inhibition

doi: 10.7554/eLife.28270

Figure Lengend Snippet: ( a ) Spatiotemporal expression patterns of SLC12A5 and members of receptor trafficking node of the KCC2 interactome in the human brain; RNAseq data were analyzed in hippocampus. Pcw, postconceptual weeks. ( b ) Native KCC2 complexes from C 12 E 9 -solubilized whole-brain membrane fractions immunoprecipitated with IgY or anti-N-term KCC2 (left) and IgG or anti-PACSIN1 (right), and immunoblotted with antibodies as indicated. ( c ) Western blot analysis of developmental expression patterns of KCC2 and PACSIN1. ( d ) Antibody-shift assay followed by 2D-BN-PAGE separation using whole-brain membrane fractions, incubated with antibodies indicated on left; gel separations were immunoblotted with anti-KCC2 or PACSIN1 antibodies. ( e ) Coimmunoprecipitation performed in COS7 cells transfected with myc-tagged KCC2b and GFP-tagged PACSIN1/2/3 constructs, immunoprecipitated with anti-N-term KCC2b, and immunoblotted with the antibodies indicated at right. ( f ) Immunoblot of immunoprecipitates from transfected COS7 cell lysates. * indicate the lanes where PACSIN1 lacks the variable region between ~aa325-383. # of independent biological replicates are indicated in parenthesis: (4), (3), (2). 10.7554/eLife.28270.025 Figure 5—source data 1. Human brain RNAseq data from the Allan Brain Atlas for receptor trafficking node.

Article Snippet: Cell lysates or solubilized membrane fractions (∼0.2–0.5 mg protein) were incubated with N-terminal KCC2b or anti-myc (CST #9B11, RRID AB_331783 ) antibodies on a rotating platform (4 hr, 4°C).

Techniques: Expressing, Membrane, Immunoprecipitation, Western Blot, Shift Assay, Incubation, Transfection, Construct