anti ap2a1  (Proteintech)

Journal: iScience

Article Title: FMRP-dependent translational control negatively regulates adapter protein complex 2-mediated endocytosis

doi: 10.1016/j.isci.2025.113062

Figure Lengend Snippet: FMRP negatively regulates the subcellular expression of AP-2 subunits (A) The subunits of three adapter protein complexes (AP1G1, AP2B1, and AP3D1), a postsynaptic marker (PSD95), a glial cell marker (GFAP), and GAPDH were examined in whole mouse brain lysates (INP) and synaptoneurosome (SNS) samples from mouse brain (postnatal day one). (B) Expression of PSD95, GFAP, and AP complexes in input (INP) and synaptoneurosomes (SNS). Signals were normalized to GAPDH. (C) AP-2 subunits, AP2A1 and AP2B1, in SNS samples prepared from eight pairs of WT and Fmr1 KO mouse brains are shown by western blot. (D) Expression levels of AP2A1 and AP2B1 in SNS samples from WT and Fmr1 KO mice. Data are represented as mean ± SEM. Unpaired t-test, N = 8, ∗ p < 0.05 and ∗∗ p < 0.01. (E) Representative images of cultured mouse cortical neurons (DIV12) from WT or Fmr1 KO mice. Immunofluorescence for AP2B1 overlaid with PSD95 and MAP2 to show the dendritic and synapse morphology. Enlarged boxes show dendritic segments of selected neurons. Scale bars, 40 μm. (F) Quantitative analysis of AP2B1 intensity and postsynaptic localization in soma and dendritic regions. Data are represented as mean ± SEM. Paired t-test, N = 3, ∗ p < 0.05 and ∗∗∗ p < 0.001.

Article Snippet: The following FAM probes: Ap2a1 (ThermoFisher Mm00475919_m1), or Ap2b1 (ThermoFisher Mm00551136_m1), were combined with VIC probe Gapdh (ThermoFisher 4326317E).

Techniques: Expressing, Marker, Western Blot, Cell Culture, Immunofluorescence

Journal: iScience

Article Title: FMRP-dependent translational control negatively regulates adapter protein complex 2-mediated endocytosis

doi: 10.1016/j.isci.2025.113062

Figure Lengend Snippet: FMRP associates with Ap2a1 and Ap2b1 mRNAs and represses nascent protein translation (A) mRNA levels of Ap2a1 and Ap2b1 in WT and Fmr1 KO mouse brains. Gapdh mRNA was used as internal control. Data are represented as mean ± SEM. Unpaired t-test, N = 8. (B) FMRP eCLIP binding sites within the mRNA sequences of Ap2a1 and Ap2b1 in E13.5 mouse brains. The intact mRNA sequences of Ap2a1 and Ap2b1 , including their 3′UTR regions, are presented, with peaks indicating the read densities of predicted binding sites within these regions. Size-matched input (SMI) represents the background reading. (C) RNA-immunoprecipitation (RIP) of FMRP-mRNA complex from mouse brain lysates. Confirmation of FMRP immunoprecipitation by western blot. (D) qPCR analysis of FMRP-mRNA complexes. Both Ap2a1 and Ap2b1 mRNAs were detected by qPCR. Psd95 and Map1b mRNAs were used as positive controls and Gapdh mRNA as a negative control. Data are represented as mean ± SEM. Paired t-test, N = 3, ∗∗∗∗ p < 0.0001. (E) Newly synthesized AP2B1 was detected by puro-PLA. WT or Fmr1 KO cortical neurons were treated with puromycin for 5 min before fixation, and the puro-PLA signal show nascent synthesis of AP2B1 within the 5 min time frame. Scale bars, 20 μm. (F) Nascent synthesis of AP2B1 in the soma (left) and dendritic (right) regions of WT and Fmr1 KO neurons. Data are represented as mean ± SEM. Paired t - test, N = 4, ∗ p < 0.05.

Article Snippet: The following FAM probes: Ap2a1 (ThermoFisher Mm00475919_m1), or Ap2b1 (ThermoFisher Mm00551136_m1), were combined with VIC probe Gapdh (ThermoFisher 4326317E).

Techniques: Control, Binding Assay, RNA Immunoprecipitation, Immunoprecipitation, Western Blot, Negative Control, Synthesized

Journal: iScience

Article Title: The adaptor protein 2 (AP2) complex modulates habituation and behavioral selection across multiple pathways and time windows

doi: 10.1016/j.isci.2024.109455

Figure Lengend Snippet: Diverse ap2s1 alleles disrupt acoustically evoked habituation learning, responsiveness, and behavior selection bias (A) Diagram of AP2 complex, annotated with the zebrafish genes encoding each subunit. The heterotetrametric complex is composed of AP2α ( ap2a1 , green), AP2β ( ap2b1 , dark gray), AP2σ ( ap2s1 , red), and AP2μ ( ap2m1a, ap2m1b , light gray). (B) Diagram of ap2s1 transcript and alleles, regions contributing to AP2 target binding indicated in orange with mutant disruptions indicated in red. (C‒H) Acoustic SLC habituation of wild type sibling (WT, blue), heterozygote sibling (Het, gray) and ap2s1 mutants (red), presented with 10 intense (23.8 dB) stimuli at 20s ISI, followed by 30 more identical stimuli at 1s ISI. Individual habituation scores were normalized to their baseline SLC response rate at 20s ISI (gray shading), and ap2s1 mutants were compared to siblings with a 2-way repeated measures ANOVA (Genotype: p < 0.0001, Time: p < 0.0001, Individual: p < 0.0001), and significant pairwise comparisons (Dunnett’s test) vs. wild type are indicated, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. Average SLC habituation scores across 300 identical stimuli at 1s ISI with hyperbolic fitted curves (F–H). (I‒K) Average responsiveness of individual larvae to 10 low intensity (−0.6 dB) acoustic stimuli presented at 20 s ISI, compared with two-tailed Mann-Whitney test with Bonferroni correction for multiple comparisons. (L‒N) Relative behavioral bias of individual larvae to 10 low intensity (−0.6 dB) acoustic stimuli presented at 20 s ISI, compared with two-tailed Mann-Whitney test with Bonferroni correction for multiple comparisons. Mutants (red) and heterozygotes (gray) were always compared to their corresponding wild type (blue) siblings, using ap2s1 p172 (C, F, I, L), ap2s1 hv1 (D, G, J, M), and ap2s1 p199 (E, H, K, N) alleles. Data are represented as mean ± SEM in all panels and n indicates individual larvae. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Article Snippet: PCR reactions were supplemented with 5% DMSO for ap2s1 p172 . ap2s1 p172 alleles were distinguished by gain of a BsmAI (NEB) cutting site, ap2s1 p199 and ap2s1 hv1 alleles were distinguished by loss of a MscI (NEB) cutting site, and ap2a1 sa1907 alleles were distinguished by gain of a HinfI (NEB) cutting site.

Techniques: Selection, Binding Assay, Mutagenesis, Two Tailed Test, MANN-WHITNEY

Journal: iScience

Article Title: The adaptor protein 2 (AP2) complex modulates habituation and behavioral selection across multiple pathways and time windows

doi: 10.1016/j.isci.2024.109455

Figure Lengend Snippet: The AP2α subunit regulates acoustically evoked escape behavior (A) Diagram of ap2a1 transcript and disrupted splice donor site of ap2a1 sa1907 (green). Core domain represented in orange and flexible appendage domain represented in blue. (B‒D) Acoustically evoked response modulation of wild types (blue, n = 36), heterozygotes (gray, n = 99) and ap2a1 sa1907 mutants (green, n = 38). Average SLC habituation scores across 30 stimuli at 1s ISI (B), normalized to each individual’s baseline SLC response levels to 10 strong (23.8 dB) stimuli at 20s ISI. ap2a1 sa1907 mutants were compared to siblings with a 2-way repeated measures ANOVA (Genotype: p < 0.0001, Time: p < 0.0001, Individual: p < 0.0001), and significant pairwise comparisons (Dunnett’s test) vs. wild type are indicated, ∗∗∗∗p < 0.0001. Average responsiveness (C) and relative behavioral bias (D) of individuals to 10 weak (−6.7 dB) and 10 strong (23.8 dB) stimuli at 20 s ISI. ∗p < 0.05, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, two-tailed Mann-Whitney test with Bonferroni correction for multiple comparisons. (E‒I) Average kinematic parameters of individual sibling (blue, n = 142) and ap2a1 sa1907 mutant (green, n = 37) larvae in response to 40 strong (23.8 dB) acoustic stimuli, separated by SLC (left) and LLC (right) responses. Parameters measured were the initial response latency (E-F), maximum turning angle of the initial C-bend (G), initial C-bend duration (H), and maximum angular velocity of the initial C-bend (I). ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, two-tailed t-tests with Welch’s correction. Data are represented as mean ± SEM in all panels.

Article Snippet: PCR reactions were supplemented with 5% DMSO for ap2s1 p172 . ap2s1 p172 alleles were distinguished by gain of a BsmAI (NEB) cutting site, ap2s1 p199 and ap2s1 hv1 alleles were distinguished by loss of a MscI (NEB) cutting site, and ap2a1 sa1907 alleles were distinguished by gain of a HinfI (NEB) cutting site.

Techniques: Two Tailed Test, MANN-WHITNEY, Mutagenesis

Journal: iScience

Article Title: The adaptor protein 2 (AP2) complex modulates habituation and behavioral selection across multiple pathways and time windows

doi: 10.1016/j.isci.2024.109455

Figure Lengend Snippet:

Article Snippet: PCR reactions were supplemented with 5% DMSO for ap2s1 p172 . ap2s1 p172 alleles were distinguished by gain of a BsmAI (NEB) cutting site, ap2s1 p199 and ap2s1 hv1 alleles were distinguished by loss of a MscI (NEB) cutting site, and ap2a1 sa1907 alleles were distinguished by gain of a HinfI (NEB) cutting site.

Techniques: Virus, Recombinant, Viscosity, Clone Assay, Plasmid Preparation, Software, Imaging

Journal: The Journal of biological chemistry

Article Title: The GPCR adaptor protein Norbin controls the trafficking of C5aR1 and CXCR4 in mouse neutrophils.

doi: 10.1016/j.jbc.2024.107940

Figure Lengend Snippet: Figure 6. Norbin is required for sustained b-arrestin recruitment and promotes the agonist-induced internalization of C5aR1 in a b-arrestin- dependent manner. A, b-arrestin recruitment. Purified Ncdnfl/fl(black) and NcdnDmye (red) neutrophils were incubated for 10 min at 37 C while being stimulated with 15 nM C5a for 0, 0.17, 1 or 10 min, as indicated. Cells were lysed, a post-granule supernatant (PGS) was prepared by centrifugation, and glycosylated proteins, including C5aR1, were purified from the PGS using wheat-germ agglutinin agarose. Proteins were analyzed by SDS-PAGE and western blotting with C5aR1 and b-arrestin (b-Arr) antibodies. Representative western blots are shown. Blots were quantified by Fiji densitometry, and the b-arrestin signal was normalized to the amount of C5aR1 in each lane. Data are mean ± SEM of five independent experiments; each dot is the mean of one experiment. Statistics are two-way ANOVA with Sidák’s multiple comparisons test. p-values in black show significant differences, p-values in gray are not significant. B, b-arrestin-dependent agonist-induced internalization. Bone marrow cells were pretreated with 100 mM barbadin for 30 min at 37 C (filled symbols), or mock-treated with 1% DMSO (open symbols), and stimulated for 5 min with 50 nM C5a, or mock-stimulated (control). Cells were stained on ice to identify live neutrophils (Mac1hi, Ly6Ghi, FVDlo) and quantify the level of C5aR1 on the neutrophil surface by flow cytometry. The mfiof C5aR1 and Mac1 on the neutrophil surface were analyzed using FlowJo. Data are mean ± SEM of 3 independent experiments; each dot is the mean of one experiment. Statistics are two-way ANOVA with Sidák’s multiple comparisons test. p-values in black show significant differences, p-values in gray are not significant. C, barbadin blocks the recruitment of the clathrin-adaptor AP2. Purified Ncdnfl/flneutrophils were incubated for 30 min at 37 C in the presence of 100 mM barbadin or mock-treated, prior to stimulation with 15 nM C5a for 1 min at 37 C. Glycosylated proteins purified from the PGS using wheat-germ agglutinin agarose as in (A) and analyzed by western blotting with AP2a1 antibody. Coomassie staining was used as a loading control. The blot shown is representative of two independent experiments. D, receptor degradation. Purified neutrophils were incubated for 2 h at 37 C while being stimulated with 15 nM C5a for the indicated periods of time towards the end. Cells were fixed, permeabilized, and stained to analyse the total cellular level of C5aR1 by flow cytometry. Left-hand panel: The mfiof total neutrophil C5aR1 was analyzed using FlowJo. Right-hand panel: AUC of the 2 h time course. Data are mean ± SEM of three independent experiments; each dot is the mean of one experiment. Statistics are two-tailed paired Student’s t test. p-values in gray are not significant.

Article Snippet: Beads were washed 3 × with 1 ml lysis buffer, and proteins were eluted by incubation in 1.3 × SDS-PAGE sample buffer at 50 C for 20 min. Proteins were analyzed by SDS-PAGE and western blotting with C5aR1, b-arrestin 1/2 (Cell Signaling Technologies, 4674, 1:500) and AP2a1 (Proteintech, 29887-1- AP, 1:1000) antibodies.

Techniques: Incubation, Centrifugation, SDS Page, Western Blot, Control, Staining, Cytometry, Two Tailed Test