Journal: iScience

Article Title: Prkn knockout mice show autistic-like behaviors and aberrant synapse formation

doi: 10.1016/j.isci.2022.104573

Figure Lengend Snippet:

Article Snippet: Rabbit anti-GluA3 (1:2000) , Cell Signaling Technology , Cat. #5117S; RRID: AB_10544796.

Techniques: Software

Journal: Frontiers in Aging Neuroscience

Article Title: Detrimental effects of soluble α-synuclein oligomers at excitatory glutamatergic synapses

doi: 10.3389/fnagi.2023.1152065

Figure Lengend Snippet: Effects of in vivo striatal injection of αSyn soluble oligomers in mice on ERK and CREB signaling. (A) Levels of pERK were evaluated by Western blot in striatal homogenates of sOligo- and PBS-injected mice 84 dpi. pERK level was normalized on total ERK expression and reported as OD% of PBS-mice. n = 9–10 mice. (B) Levels of pCREB were evaluated by Western blot in striatal homogenates of sOligo- and PBS-injected mice 84 dpi. pCREB level was normalized on total CREB expression and reported as OD% of PBS-mice. n = 10–11 mice. Data are represented as mean ± SEM. * P < 0.05 (Student’s t -test; data with non-normal distribution were tested with Mann–Whitney test).

Article Snippet: The primary antibodies used were: mouse anti-tubulin (1:30,000, #T9026, Sigma), rabbit anti-GAPDH (1:5,000, #sc-25778, Santa Cruz), rabbit anti-GluN2A (1:1,000, #M264, Sigma), rabbit anti-GluN2B (1:1,000, #718600, Invitrogen), mouse anti-GluN2D (1:1,000, #MAB5578, Millipore), rabbit anti-GluA1 (1:1,000, #13185, Cell Signaling), mouse anti-GluA2 (1:1,000, #75–002, Neuromab), mouse anti-GluA3 (1:1,000, #MAB5416, Millipore), polyclonal anti-Rph3A (1:2,000, Protein Tech, #11396-1-AP), mouse anti-PSD-95 (1:1,000, #K28/43, Neuromab), rabbit anti-tyrosine hydroxylase (1:10,000, #AB152, Millipore), rabbit anti-phospho-extracellular signal-regulated kinase (ERK) 44/42 (1:1,000, Cell Signaling, #9101), rabbit anti-ERK 44/42 (1:1,000, #9102, Cell Signaling), rabbit anti-phospho-cAMP responsive element binding protein (CREB) (1:1,000, #9198, Cell Signaling), and rabbit anti-CREB (1:1,000, #9197, Cell Signaling).

Techniques: In Vivo, Injection, Western Blot, Expressing, MANN-WHITNEY

Journal: Frontiers in Aging Neuroscience

Article Title: Detrimental effects of soluble α-synuclein oligomers at excitatory glutamatergic synapses

doi: 10.3389/fnagi.2023.1152065

Figure Lengend Snippet: Molecular effects induced by αsyn sOligo at the hippocampal synapse in vitro . Primary cultures of rat hippocampal neurons were treated at DIV9 with either 2 μg/μl sOligo or PBS and analyses were performed at DIV16. Post-synaptic levels of (A) AMPAR (GluA1, GluA2, and GluA3) and NMDAR (GluN2A and GluN2B) subunits and (B) the scaffolding protein Rph3A and PSD-95 were evaluated by Western blot in TIF from sOligo- or PBS-neurons. Protein levels normalized on tubulin were reported as OD% of PBS-neurons. n = 6 independent cultures. (C) Representative confocal images and quantification of spine morphology analyses (spine density, spine length and spine width) of sOligo- and PBS-neurons at DIV16. Scale bar: 3 μm. n = 23–24 neurons from 3 independent cultures. (D) Levels of pERK were evaluated by Western blot in homogenates of sOligo- and PBS-neurons at DIV16. pERK level was normalized on total ERK expression and reported as OD% of PBS-neurons. n = 12–13 independent cultures. (E) Levels of pCREB were evaluated by Western blot in homogenates of sOligo- and PBS-neurons at DIV16. pCREB level was normalized on total CREB expression and reported as OD% of PBS-neurons. n = 14 independent cultures. Data are represented as mean ± SEM. * P < 0.05 (Student’s t -test; data with non-normal distribution were tested with Mann–Whitney test).

Article Snippet: The primary antibodies used were: mouse anti-tubulin (1:30,000, #T9026, Sigma), rabbit anti-GAPDH (1:5,000, #sc-25778, Santa Cruz), rabbit anti-GluN2A (1:1,000, #M264, Sigma), rabbit anti-GluN2B (1:1,000, #718600, Invitrogen), mouse anti-GluN2D (1:1,000, #MAB5578, Millipore), rabbit anti-GluA1 (1:1,000, #13185, Cell Signaling), mouse anti-GluA2 (1:1,000, #75–002, Neuromab), mouse anti-GluA3 (1:1,000, #MAB5416, Millipore), polyclonal anti-Rph3A (1:2,000, Protein Tech, #11396-1-AP), mouse anti-PSD-95 (1:1,000, #K28/43, Neuromab), rabbit anti-tyrosine hydroxylase (1:10,000, #AB152, Millipore), rabbit anti-phospho-extracellular signal-regulated kinase (ERK) 44/42 (1:1,000, Cell Signaling, #9101), rabbit anti-ERK 44/42 (1:1,000, #9102, Cell Signaling), rabbit anti-phospho-cAMP responsive element binding protein (CREB) (1:1,000, #9198, Cell Signaling), and rabbit anti-CREB (1:1,000, #9197, Cell Signaling).

Techniques: In Vitro, Scaffolding, Western Blot, Expressing, MANN-WHITNEY

Journal: PLoS Biology

Article Title: Adult medial habenula neurons require GDNF receptor GFRα1 for synaptic stability and function

doi: 10.1371/journal.pbio.3001350

Figure Lengend Snippet: (A, B) Immunoblots of synaptic protein fractions from mHb (A) and IPN (B) of WT, Het, and KO mice sacrificed 1 month after tamoxifen treatment probed for GluA1, 2, 3 and 4 as indicated. PSD95 was probed as loading control. Quantifications (± SEM) of GluA1 to 4 levels were corrected for PSD95 levels and normalized to levels in WT samples. N = 9–10 samples per group; 1-way ANOVA analysis followed by Tukey multiple comparison test; * P < 0.05; ** P < 0.01. (C, E) Immunoblots of synaptic protein fractions from mHb (C) and IPN (E) of WT, Het, and KO mice probed for GluA1 P-Ser 831 and P-Ser 845 as indicated and reported for total GluA1 and Tubulin as loading controls. (D, F) Quantifications (± SEM) of GluA1 P-Ser 831 and P-Ser 845 levels in mHb (D, N = 13 samples per group) and IPN (F, N = 14 samples per group) corrected for total GluA1 levels and normalized to levels in WT samples. Student t test; * P < 0.05. (G, I) PLA signals (red) for GluA1-GluA2 (G) and GluA1-GluA4 (I) complexes in coronal sections of mHb and core IPN from WT and KO mice. Counterstaining with DAPI appears in blue. Scale bars, 20 μm. (H, J) Quantification (± SEM) of PLA puncta for GluA1-GluA2 (H) and GluA1-GluA4 (J) complexes in mHb, core IPN and lateral IPN (IPL) from WT and KO mice ( N = 5 mice per group). A total of 16 images from the mHb (8 dorsal and 8 ventral) and 18 images from the IPN (core IPN: 6 dorsal and 6 ventral; 6 lateral) were analyzed per mouse. Student t test; * P < 0.05. The data underlying this figure can be found at https://figshare.com/projects/Raw_Data_Fernandez-Suarez_et_al_2021/123406 . GFRα1, glial cell–derived neurotrophic factor receptor alpha 1; HET, heterozygous; IPN, interpeduncular nucleus; KO, knockout; mHb, medial habenula; PLA, proximity ligation assay; PSD, postsynaptic density; WT, wild-type.

Article Snippet: The following primary antibodies were used at the indicated dilutions: Goat anti GFRα1 (1:500, AF560, RnD, USA): goat anti c-RET (1:500, AF482, RnD); rabbit anti NCAM (1:1,000, AB5032, Millipore, USA); Mouse anti PSD95 (1:1,000, MA1-046, Thermo Scientific); rabbit anti GluA1 (1:1,000, AB1504, Millipore); rabbit anti GluA2 (1:1,000, AB1768-I, Millipore); rabbit anti GluA3 (1:1,000, AGC-010, Alomone Labs, Israel); rabbit anti GluA4 (1:1,000, AB1508, Millipore); rabbit anti phospho-Ser831 GluA1 (1:500, 75574, Cell Signaling, USA); rabbit anti phospho-Ser845 GluA1 (1:500, 8084, Cell Signaling); and mouse anti tubulin alpha (1:10,000, T6199, Sigma-Aldrich).

Techniques: Western Blot, Control, Comparison, Derivative Assay, Knock-Out, Proximity Ligation Assay

Journal: PLoS Biology

Article Title: Adult medial habenula neurons require GDNF receptor GFRα1 for synaptic stability and function

doi: 10.1371/journal.pbio.3001350

Figure Lengend Snippet: (A, B) Immunoblots of synaptic protein fractions from mHb (A) and IPN (B) of WT, Het, and KO mice sacrificed 1 month after tamoxifen treatment probed for GluA1, 2, 3 and 4 as indicated. PSD95 was probed as loading control. Quantifications (± SEM) of GluA1 to 4 levels were corrected for PSD95 levels and normalized to levels in WT samples. N = 9–10 samples per group; 1-way ANOVA analysis followed by Tukey multiple comparison test; * P < 0.05; ** P < 0.01. (C, E) Immunoblots of synaptic protein fractions from mHb (C) and IPN (E) of WT, Het, and KO mice probed for GluA1 P-Ser 831 and P-Ser 845 as indicated and reported for total GluA1 and Tubulin as loading controls. (D, F) Quantifications (± SEM) of GluA1 P-Ser 831 and P-Ser 845 levels in mHb (D, N = 13 samples per group) and IPN (F, N = 14 samples per group) corrected for total GluA1 levels and normalized to levels in WT samples. Student t test; * P < 0.05. (G, I) PLA signals (red) for GluA1-GluA2 (G) and GluA1-GluA4 (I) complexes in coronal sections of mHb and core IPN from WT and KO mice. Counterstaining with DAPI appears in blue. Scale bars, 20 μm. (H, J) Quantification (± SEM) of PLA puncta for GluA1-GluA2 (H) and GluA1-GluA4 (J) complexes in mHb, core IPN and lateral IPN (IPL) from WT and KO mice ( N = 5 mice per group). A total of 16 images from the mHb (8 dorsal and 8 ventral) and 18 images from the IPN (core IPN: 6 dorsal and 6 ventral; 6 lateral) were analyzed per mouse. Student t test; * P < 0.05. The data underlying this figure can be found at https://figshare.com/projects/Raw_Data_Fernandez-Suarez_et_al_2021/123406 . GFRα1, glial cell–derived neurotrophic factor receptor alpha 1; HET, heterozygous; IPN, interpeduncular nucleus; KO, knockout; mHb, medial habenula; PLA, proximity ligation assay; PSD, postsynaptic density; WT, wild-type.

Article Snippet: The following primary antibodies were used at the indicated dilutions: Goat anti GFRα1 (1:500, AF560, RnD, USA): goat anti c-RET (1:500, AF482, RnD); rabbit anti NCAM (1:1,000, AB5032, Millipore, USA); Mouse anti PSD95 (1:1,000, MA1-046, Thermo Scientific); rabbit anti GluA1 (1:1,000, AB1504, Millipore); rabbit anti GluA2 (1:1,000, AB1768-I, Millipore); rabbit anti GluA3 (1:1,000, AGC-010, Alomone Labs, Israel); rabbit anti GluA4 (1:1,000, AB1508, Millipore); rabbit anti phospho-Ser831 GluA1 (1:500, 75574, Cell Signaling, USA); rabbit anti phospho-Ser845 GluA1 (1:500, 8084, Cell Signaling); and mouse anti tubulin alpha (1:10,000, T6199, Sigma-Aldrich).

Techniques: Western Blot, Control, Comparison, Derivative Assay, Knock-Out, Proximity Ligation Assay