Journal: iScience

Article Title: Proximity interactome mapping furthers a role for spinophilin in protein homeostasis

doi: 10.1016/j.isci.2025.113735

Figure Lengend Snippet: Generation and validation of a Cre-dependent spinophilin-ALFA-UltraID construct (A) Map of the sequence validated spinophilin-ALFA-UltraID construct created in SnapGene. (B–F and H) HEK293 cells were transfected with the spinophilin-ALFA-UltraID construct (Spinophilin-UID) along with a construct encoding an improved Cre recombinase (Cre) or an empty pDonr221 vector (pDonr). Following transfections, cells were incubated with biotin. HEK293 lysates were then precipitated using streptavidin magnetic beads (Str) or neutravidin agarose beads (Neu) (B) Neu agarose beads or trypsin-resistant streptavidin magnetic beads (MS) (C–F and H). Inputs (Inp), post-pull-down lysates (depleted), and pull-downs were separated by SDS-PAGE and probed with an Alexa Dye 800-conjugated streptavidin protein or immunoblotted with an ALFA-tag (Alfa), spinophilin, PP1α, or D2R antibody. Blots were imaged using a LiCor Odyssey M. (G) The total protein, streptavidin, spinophilin, or PP1α signal intensity in the depleted lysate was divided by the signal intensity in the input to determine the efficiency of Neu pull-down. Data are represented as mean ± SEM.

Article Snippet: PP1α (E-9) Mouse , Santa Cruz , SC-7482, RRID: AB_628177.

Techniques: Biomarker Discovery, Construct, Sequencing, Transfection, Plasmid Preparation, Incubation, Magnetic Beads, SDS Page

Journal: iScience

Article Title: Proximity interactome mapping furthers a role for spinophilin in protein homeostasis

doi: 10.1016/j.isci.2025.113735

Figure Lengend Snippet: Comparison of neutravidin pull-down and ALFA-tag nanobody immunoprecipitation to trypsin-resistant streptavidin beads HEK293 cells were transfected with the spinophilin-ALFA-UltraID construct (Spinophilin-UID) along with a construct encoding an improved Cre recombinase (Cre) or were non-transfected. (A) Following transfections, cells were incubated with biotin. HEK293 lysates were then precipitated using neutravidin agarose beads (Neu) or trypsin-resistant streptavidin magnetic beads (MS). Inputs (I) and pull-downs (PD) were separated by SDS-PAGE and imaged for total protein with Revert stain and subsequently immunoblotted for spinophilin, PP1α, Bip, beta-tubulin (tubulin), Rps3, or Hsc70. Blots were imaged using a LiCor Odyssey M. (B) Following transfections, cells were incubated with biotin. HEK293 lysates were then precipitated using neutravidin agarose beads (Neu) or an ALFA-tag nanobody fused to agarose beads (AL). Inputs (I) and pull-downs (PD) were separated by SDS-PAGE and imaged for total protein with Revert stain and subsequently immunoblotted for spinophilin, PP1α, Bip, beta-tubulin (tubulin), Rps3, or Hsc70. Blots were imaged using a LiCor Odyssey M.

Article Snippet: PP1α (E-9) Mouse , Santa Cruz , SC-7482, RRID: AB_628177.

Techniques: Comparison, Immunoprecipitation, Transfection, Construct, Incubation, Magnetic Beads, SDS Page, Staining

Journal: iScience

Article Title: Proximity interactome mapping furthers a role for spinophilin in protein homeostasis

doi: 10.1016/j.isci.2025.113735

Figure Lengend Snippet: Unique subcellular distribution of spinophilin (Ppp1r9b) and neurabin (Ppp1r9a) mRNA and a role for spinophilin in regulating its own expression (A and B) RNAScope analysis of WT mouse brains at 4X stitched together (A) and 63× magnification (B). Scale bar lengths represent 3 mm (A) and 25 μm (B). (C) Spinophilin fl/fl /A2A control mice (Spino fl/fl or mice expressing AdorA2A-Cre [A2A] or Drd1-Cre [D1]) or mice with loss of spinophilin in iMSNs (Spinophilin fl/fl /A2A) or dMSNs (Spinophilin fl/fl /D1) were transduced with a virus encoding a Cre-dependent, HA-tagged spinophilin. Total lysates (input) were immunoblotted for HA epitope tag and PP1α and HA immunoprecipitates were immunoblotted for spinophilin and PP1α.

Article Snippet: PP1α (E-9) Mouse , Santa Cruz , SC-7482, RRID: AB_628177.

Techniques: Expressing, RNAscope, Control, Transduction, Virus

Journal: iScience

Article Title: Proximity interactome mapping furthers a role for spinophilin in protein homeostasis

doi: 10.1016/j.isci.2025.113735

Figure Lengend Snippet: Spinophilin functions to stabilize PSD proteins Striata from wild-type (WT) or spinophilin knockout (KO) mice were homogenized and biochemically fractionated into synaptoneurosome (Syn) or postsynaptic density (PSD) fractions. (A) Total homogenate (Hom), Syn, and PSD fractions were separated by SDS page and stained with Revert total protein stain or immunoblotted for spinophilin, PSD95, GAPDH, GluN2B, GluA2, PP1α, and PP1γ1. (B) The fluorescence intensity of the specific protein in the homogenate, Syn, or PSD fraction from spinophilin WT mice was normalized to the total protein abundance within the corresponding fraction. A ratio was generated by dividing the Syn or PSD fraction values by the homogenate values. (C and D) The fluorescence intensity of the specific protein in the Syn (C) or PSD (D) fraction isolated from spinophilin WT or KO mice was divided by the fluorescence intensity of the protein within the corresponding homogenate fraction. A ratio was generated by dividing the KO value by the WT value. (E) The fluorescence intensity of the total protein stain in the Syn or PSD fraction isolated from spinophilin WT or KO mice was divided by the fluorescence intensity of the total protein stain within the corresponding homogenate fraction. A ratio was generated by dividing the KO value by the WT value. (F–H) The fluorescence intensity of the specific protein in the homogenate (F), Syn (G), and PSD (H) fraction isolated from spinophilin WT or KO mice was divided by the fluorescence intensity of the total protein stain within the corresponding fraction. A ratio was generated by dividing the KO value by the WT value. (I and J) Total homogenate (H), Syn (S), and/or PSD (P) fractions were separated by SDS page and immunoblotted for RPS6 or pRPS6 (Ser240/244). See and for all full blots used for quantitation. Individual data points ( n = 4) and plotted as mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001; one sample t test (B–H), unpaired t test (I and J). See for all statistics analyses.

Article Snippet: PP1α (E-9) Mouse , Santa Cruz , SC-7482, RRID: AB_628177.

Techniques: Knock-Out, SDS Page, Staining, Fluorescence, Quantitative Proteomics, Generated, Isolation, Quantitation Assay