Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: The STEP 61 interactome reveals subunit-specific AMPA receptor binding and synaptic regulation

doi: 10.1073/pnas.1900878116

Figure Lengend Snippet: AMPARs and NMDARs are differentially regulated in STEP-KO mouse brain. (A) Using adult WT and STEP-KO mouse forebrain, a subcellular fractionation assay was performed as described in Materials and Methods, and lysates were immunoblotted with indicated antibodies. H, homogenate; S1, supernatant; Tx-100 sol., soluble fraction. (B–C) Quantification of blots normalized to β-actin (n = 5 independent experiments). Each band intensity in homogenate (total) using GluA1 (P = 0.066), GluA2 (P = 0.006), GluA3 (P = 0.026), GluN2A (P = 0.041), and GluN2B (P = 0.001) (B), as well as GluA1 (P = 0.339), GluA2 (P = 0.007), GluA3 (P = 0.006), GluN2A (P = 0.148), and GluN2B (P = 0.297) in PSD (synaptic) fraction (C) was measured using ImageJ software. (D) The PSD fraction was isolated from WT and STEP-KO mouse brain, solubilized with 1% SDS lysis buffer, and neutralized with 1% Triton X-100 lysis buffer, and an immunoprecipitation (IP) assay was performed using 4G10 (pan pY) antibody. Proteins were resolved by SDS/PAGE and immunoblotted with GluA2 antibody. Quantification of blots normalized to input GluA2 (n = 3 independent experiments, P = 0.007). Error bars represent +SEM, *P < 0.05, **P < 0.01, P value is compared with WT.

Article Snippet: Mouse anti-STEP (cat. no. NB300-202; Novagen), rabbit anti-SynGAP (cat. no. 3200; Cell Signaling), rabbit anti–Kalirin-7 (laboratory made), mouse anti–NLGN-1 (cat. no. 129111; Synaptic Systems), rabbit anti-NBEA (cat. no. 194003; Synaptic Systems), rabbit anti-GluA1 (custom made), rabbit anti-GluA2/3 ( 55 ), mouse anti-GluA2 (clone L21/32; NeuroMab), rabbit anti-GluA2 (cat. 82103; Synaptic Systems), rabbit anti-GluA3 (cat. no. 3437; Cell Signaling), rabbit anti-GluN2A (cat. no. M264; Sigma), mouse anti-GluN2B (clone N59/36; NeuroMab), mouse anti-GluN1 ( 28 ), mouse anti–PSD-95 (clone K28/43; NeuroMab), mouse anti-SAP102 (clone N19/2; NeuroMab), rabbit anti-Fyn (cat. no. 4023; Cell Signaling), rabbit anti-Src (cat. no. 2110; Cell Signaling), mouse anti-GAPDH (cat. no. sc-365062; Santa Cruz), mouse anti-FLAG (cat. no. F1804; Sigma), rabbit anti-FLAG (cat. no. 7425; Sigma), rabbit anti-HA (cat. no. 3724; Cell Signaling), mouse anti-HA (cat. no. 2367; Cell Signaling), rabbit anti-myc (cat. no. 2278; Cell Signaling), rabbit anti-GST (cat. no. A190-122A; Bethyl Laboratories), mouse anti–β-actin (cat. no. G043; abm), and mouse anti-4G10 (cat. no. 05-321; Millipore).

Techniques: Fractionation, Software, Isolation, Lysis, Immunoprecipitation, SDS Page

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: The STEP 61 interactome reveals subunit-specific AMPA receptor binding and synaptic regulation

doi: 10.1073/pnas.1900878116

Figure Lengend Snippet: STEP61 overexpression regulates the synaptic expression of AMPARs and NMDARs, and STEP61 regulation of synaptic AMPARs is through the lysosomal degradation pathway. (A–C) Primary cortical neurons were transduced with lentivirus expressing STEP61 at DIV 17, and 7 d later, total protein lysate (n = 5 independent experiments) or PSD fraction (n = 3 independent experiments) was isolated and immunoblotted with GluA1 (P = 0.072), GluA2 (P = 0.227), GluA3 (P = 0.306), GluN2A (P = 0.172), GluN2B (P = 0.479), GluN1 (P = 0.483), and Fyn (P = 0.357) in total lysate (A), as well as with GluA1 (P = 0.421), GluA2 (P = 0.002), GluA3 (P = 3.628e-04), GluN2A (P = 0.009), GluN2B (P = 0.001), GluN1 (P = 3.077e-05), and Fyn (P = 0.173) in PSD fraction (B). (C) Chloroquine (50 μM) or MG-132 (1 μM) was applied for 18 h before protein isolation from the PSD fraction. Immunoblotting was performed (n = 3 independent experiments) with GluA1 (STEP61, P = 0.7360; STEP61 + chloroquine, P = 0.9645; and STEP61 + MG-132, P = 0.9278), GluA2 (STEP61, P = 0.0116; STEP61 + chloroquine, P = 0.9979; and STEP61 + MG-132, P = 0029), and GluA3 (STEP61, P = 0.0001; STEP61 + chloroquine, P = 0.3268; and STEP61 + MG-132, P = 0.0004). All blots were normalized to β-actin. Dunnett’s one-way ANOVA test was performed. Error bars represent +SEM, *P < 0.05, **P < 0.01, ***P < 0.001, P value is compared with control (CTL). (D, Left) Primary hippocampal neurons were transfected with pCAG-IRES-mCherry or pCAG-STEP61-IRES-mCherry at DIV 10 and immunostained at DIV 20. After fixation and permeabilization, total GluA2 was labeled with anti-GluA2 and Alexa 555-conjugated secondary antibody (red), and PSD-95 was visualized with anti-PSD-95 and Alexa 488-conjugated secondary antibody (green). (Scale bar: 5 μm.). Arrowheads indicate GluA2 nonoverlapping with PSD-95 puncta. (D, Right) STEP61 (P = 0.0001), STEP61 + chloroquine (P = 0.9953), and STEP61 + MG-132 (P = 0.0001), Dunnett’s one-way ANOVA test. Error bars represent ±SEM, ***P < 0.001, P value is compared with CTL. ns, not significant.

Article Snippet: Mouse anti-STEP (cat. no. NB300-202; Novagen), rabbit anti-SynGAP (cat. no. 3200; Cell Signaling), rabbit anti–Kalirin-7 (laboratory made), mouse anti–NLGN-1 (cat. no. 129111; Synaptic Systems), rabbit anti-NBEA (cat. no. 194003; Synaptic Systems), rabbit anti-GluA1 (custom made), rabbit anti-GluA2/3 ( 55 ), mouse anti-GluA2 (clone L21/32; NeuroMab), rabbit anti-GluA2 (cat. 82103; Synaptic Systems), rabbit anti-GluA3 (cat. no. 3437; Cell Signaling), rabbit anti-GluN2A (cat. no. M264; Sigma), mouse anti-GluN2B (clone N59/36; NeuroMab), mouse anti-GluN1 ( 28 ), mouse anti–PSD-95 (clone K28/43; NeuroMab), mouse anti-SAP102 (clone N19/2; NeuroMab), rabbit anti-Fyn (cat. no. 4023; Cell Signaling), rabbit anti-Src (cat. no. 2110; Cell Signaling), mouse anti-GAPDH (cat. no. sc-365062; Santa Cruz), mouse anti-FLAG (cat. no. F1804; Sigma), rabbit anti-FLAG (cat. no. 7425; Sigma), rabbit anti-HA (cat. no. 3724; Cell Signaling), mouse anti-HA (cat. no. 2367; Cell Signaling), rabbit anti-myc (cat. no. 2278; Cell Signaling), rabbit anti-GST (cat. no. A190-122A; Bethyl Laboratories), mouse anti–β-actin (cat. no. G043; abm), and mouse anti-4G10 (cat. no. 05-321; Millipore).

Techniques: Over Expression, Expressing, Transduction, Isolation, Western Blot, Transfection, Labeling

Journal: iScience

Article Title: The decreased astrocyte-microglia interaction reflects the early characteristics of Alzheimer’s disease

doi: 10.1016/j.isci.2024.109281

Figure Lengend Snippet:

Article Snippet: Primary antibodies used were: anti-Iba1 (1:1000, rabbit, 019–19741, Wako), anti-GFAP (1:1000, rabbit, goat, ab53554, Abcam), anti-6E10 (1:1000, mouse, 803014, biolegends), anti- Synaptophysin (1:1000, rabbit, ab14692, Abcam), anti-Gria1 (1:20000, mouse, 67642-1-IG, proteintech), anti-Gria2 (1:500, rabbit, 11994-1-IG, proteintech), anti-Gria3 (1:1000, rabbit, 29588-1-IG, proteintech), anti-Gria4 (1:500, rabbit, 23350-1-IG, proteintech), anti-PSD95 (1:2000, rabbit, 20665-1-IG, proteintech), anti-Vglut1 (1:1000, rabbit, 12331, Cell signaling), anti-Vim (1:1000, rabbit, YT4880, immunoway), anti-C1qa (1:1000, rabbit, YN0612, immunoway), anti-Lcp1 (1:1000, rabbit, YN0002, immunoway), anti-Anxa3 (1:1000, rabbit, YT0237, immunoway), anti-Anxa5 (1:1000, rabbit, ab14196, Abcam), anti- Ctsb (1:1000, goat, AF965, RD), anti-β-actin (1:5000, mouse, A5441, sigma), Confocal fluorescence images were acquired using a Nikon A1 confocal laser scanning microscope with ×20 objectives for imaging stained or autofluorescent neurons.

Techniques: Virus, Bicinchoninic Acid Protein Assay, Mass Spectrometry, Software