Journal: Molecular Brain

Article Title: Protein tyrosine phosphatase receptor type R is required for Purkinje cell responsiveness in cerebellar long-term depression

doi: 10.1186/s13041-014-0092-8

Figure Lengend Snippet: ERK and MEK phosphorylation profiles following LTD induction in wild type and Ptprr −/− cerebellar slices. (A) Immunoblot detecting phospho-ERK1/2 (upper panel) and total ERK1/2 (lower panel) in cerebellar slices of wild-type and Ptprr −/− mice under basal conditions, after a 5-min stimulation with K-Glu to induce LTD, and following subsequent 10 or 20 min washing out of K-Glu. Representative images are shown. (B) Quantitative representation of phospho-ERK1/2 over total ERK1/2 levels (arbitrary units) in lysates of cerebellar slices as determined under (A). Results are presented as mean values and error bars indicate SEM from five independent experiments (Student’s t -test, *p < 0.05). (C) An alternative quantitative representation of the data determined under (A) showing the fold increase of phospho-ERK1/2-total ERK1/2 ratios in the different samples as compared to the ratio under basal conditions. Results are presented as mean values and error bars indicate SEM from five independent experiments (Student’s t -test, *p < 0.05). (D) Immunoblot detecting phospho-MEK1/2 (upper panel) and total MEK1/2 (lower panel) in cerebellar slices of wild-type and Ptprr −/− mice under basal conditions, stimulated with K-Glu to induce LTD, or after subsequent wash out of K-Glu for 10 or 20 min. Representative images out of five independent experiments are shown. (E) Quantification of the ratio of phospho-MEK1/2 over total MEK1/2 levels (arbitrary units) from the cerebellar slice lysates as described under (D). Results are presented as mean values and error bars indicate SEM from five independent experiments. (F) Quantitative representation of the fold increase of relative phospho-MEK1/2 levels compared to the relative levels under basal conditions as determined in the cerebellar slice lysates described under (D). Results are presented as mean values and error bars indicate SEM from five independent experiments (Student’s t -test, **p < 0.01).

Article Snippet: Primary antibodies (all from Cell Signaling Technology, unless otherwise specified) were rabbit polyclonal anti-ERK1/2 (#9102), mouse monoclonal anti-pT202/Y204-ERK1/2 (#9106), mouse monoclonal anti-MEK1/2 (#4694), rabbit monoclonal anti-pS217/S221-MEK1/2 (#9154), mouse monoclonal anti-GluA2 (MAB397, Merck Millipore), rabbit polyclonal anti-pS880-GluA2 (ab52180, Abcam), rabbit polyclonal anti-pY869/Y873/Y876-GluA2 (#3921), rabbit monoclonal anti-Src (#2109), rabbit monoclonal anti-pY416-Src (#6943), and rabbit monoclonal anti-GAPDH (#2118).

Techniques: Western Blot

Journal: Molecular Brain

Article Title: Protein tyrosine phosphatase receptor type R is required for Purkinje cell responsiveness in cerebellar long-term depression

doi: 10.1186/s13041-014-0092-8

Figure Lengend Snippet: ERK and MEK phosphorylation profiles following LTD induction in wild type and Ptprr −/− cerebellar slices. (A) Immunoblot detecting phospho-ERK1/2 (upper panel) and total ERK1/2 (lower panel) in cerebellar slices of wild-type and Ptprr −/− mice under basal conditions, after a 5-min stimulation with K-Glu to induce LTD, and following subsequent 10 or 20 min washing out of K-Glu. Representative images are shown. (B) Quantitative representation of phospho-ERK1/2 over total ERK1/2 levels (arbitrary units) in lysates of cerebellar slices as determined under (A). Results are presented as mean values and error bars indicate SEM from five independent experiments (Student’s t -test, *p < 0.05). (C) An alternative quantitative representation of the data determined under (A) showing the fold increase of phospho-ERK1/2-total ERK1/2 ratios in the different samples as compared to the ratio under basal conditions. Results are presented as mean values and error bars indicate SEM from five independent experiments (Student’s t -test, *p < 0.05). (D) Immunoblot detecting phospho-MEK1/2 (upper panel) and total MEK1/2 (lower panel) in cerebellar slices of wild-type and Ptprr −/− mice under basal conditions, stimulated with K-Glu to induce LTD, or after subsequent wash out of K-Glu for 10 or 20 min. Representative images out of five independent experiments are shown. (E) Quantification of the ratio of phospho-MEK1/2 over total MEK1/2 levels (arbitrary units) from the cerebellar slice lysates as described under (D). Results are presented as mean values and error bars indicate SEM from five independent experiments. (F) Quantitative representation of the fold increase of relative phospho-MEK1/2 levels compared to the relative levels under basal conditions as determined in the cerebellar slice lysates described under (D). Results are presented as mean values and error bars indicate SEM from five independent experiments (Student’s t -test, **p < 0.01).

Article Snippet: Primary antibodies (all from Cell Signaling Technology, unless otherwise specified) were rabbit polyclonal anti-ERK1/2 (#9102), mouse monoclonal anti-pT202/Y204-ERK1/2 (#9106), mouse monoclonal anti-MEK1/2 (#4694), rabbit monoclonal anti-pS217/S221-MEK1/2 (#9154), mouse monoclonal anti-GluA2 (MAB397, Merck Millipore), rabbit polyclonal anti-pS880-GluA2 (ab52180, Abcam), rabbit polyclonal anti-pY869/Y873/Y876-GluA2 (#3921), rabbit monoclonal anti-Src (#2109), rabbit monoclonal anti-pY416-Src (#6943), and rabbit monoclonal anti-GAPDH (#2118).

Techniques: Western Blot

Journal: bioRxiv

Article Title: PP1β opposes classic PP1 function, inhibiting spine maturation and promoting LTP

doi: 10.1101/2023.01.26.525737

Figure Lengend Snippet: ( A-D ) Western blot results from CA1 microdissections of PP1β cKO mice and littermate controls. 3 mice per group. Phosphorylation was normalized to total protein, and total protein was normalized to β-tubulin. ( A ) Signaling pathways relevant to synaptic plasticity. No significant differences were observed. ( B ) AMPAR phosphorylation and total protein levels. S831 GluA1 phosphorylation was significantly increased in PP1β cKO (two-tailed t-test: p<0.05). ( C ) NMDAR phosphorylation and total protein levels. Total GluN2A levels were significantly decreased (two-tailed t-test: p<0.01). ( D ) Quantification of comparisons. Following normalization to total protein or β-tubulin, intensities were normalized to control samples. ( E-G ) Results from mEPSC recordings from CA1 pyramidal neurons. ( E ) PP1β cKO has no effect on mean mEPSC amplitude (two-tailed t-test: p=0.31, N=7 (control) and 6 (cKO) mice, n=26 and 31) but alters the amplitude distribution (Kolmogorov-Smirnov: p<0.0001, 1000 randomly sampled events per group). ( F ) PP1β cKO significantly increases mEPSC frequency (two-tailed t-test: p<0.01, N=7 and 6, n=26 and 31) and correspondingly shifts the frequency distribution (Kolmogorov-Smirnov: p<0.0001, 1000 randomly sampled events per group). ( G ) Example mEPSC traces from control (top) and a PP1β cKO (bottom). ( H-J ) Spine number and morphology of CA1 pyramidal neurons. ( H ) Examples of dendritic spines in biocytin-filled CA1 pyramidal cells. ( I ) PP1β cKO significantly increases spine density (two-tailed t-test: p<0.05, N=4 (control) and 3 (cKO) mice, n=23 and 19 dendrites). ( J ) PP1β cKO alters spine morphology (two-way ANOVA, genotype: p<0.05), significantly increasing mushroom spines (Šidák, p<0.001), as categorized by Imaris reconstruction. ( K ) Examples of minimal stimulation experiments from control (top) and PP1β cKO (bottom) mice. Failures are shown in grey, successful responses in black. Insets show waveforms from averaged responses at −70 mV (top left) and +40 mV (bottom right) holding potential, scale bars 5 pA, 10 ms. Y-axis dotted lines denote 5 pA thresholds used for initial classification. X-axis dotted line denotes the switch to +40 mV holding potential. All events were manually reviewed. ( L ) PP1β cKO decreases the proportion of silent synapses in CA1 pyramidal cells (two-tailed t-test: p<0.05, N=4 and 7, n=8 and 10 cells).

Article Snippet: The following antibodies were used: β-tubulin (Cell Signaling Technology (CST), 5666), CaMKII pT286 (CST, 12716), CaMKII (Millipore, 04-1079), CREB pS133 (CST, 9191), CREB (CST, 9104), ERK (CST, 9102), GluA1 pS831 (Sigma, A4352), GluA1 pS845 (Millipore, AB5849), GluA1 (Millipore, AB1504), GluA2 pS880 (Millipore, 07-294-I), GluA2 (Millipore, MAB397), GluA2/3 (Upstate, 07-598), GluN1 (Invitrogen, 32-0500), GluN2B pS1480 (PA1-4733), GluN2B (Santa Cruz, 1469), GluN2A (Santa Cruz, 1468).

Techniques: Western Blot, Phospho-proteomics, Protein-Protein interactions, Two Tailed Test, Control