Journal: The Journal of Experimental Medicine

Article Title: Regulation of amyloid-β dynamics and pathology by the circadian clock

doi: 10.1084/jem.20172347

Figure Lengend Snippet: Intrinsic hippocampal molecular circadian clock function does not influence the daily rhythm of hippocampal ISF Aβ. (A) Schematic depicting tissue-specific AAV-Cre–mediated excision of Bmal1 within the hippocampus of Bmal1 f/f ; APPPS1-21 transgenic mice. (B) Diagram depicting Bmal1 deletion abrogating circadian clock function. In addition to Per and Cry genes, BMAL1 regulates transcription of many other clock-controlled genes in a tissue-specific manner. (C) Schematic illustrating the in vivo microdialysis method used to collect soluble Aβ in the hippocampus subsequent to viral injections. This technique was performed in awake, behaving mice. (D) Representative images of microdialysis probe placement (dashed lines) within the hippocampus of mice injected with either AAV-GFP (labeled Cre − ; APP, left) with normal BMAL1 expression (bottom left) or injected with AAV-Cre–IRES-GFP (labeled Cre + ; APP, right), resulting in a loss of local hippocampal BMAL1 expression (bottom right). Bar, 1 mm. (E) Graph showing that hourly ISF Aβ levels are not influenced by the targeted hippocampal excision of Bmal1 (green, AAV-Cre–labeled Cre + ; APP) compared with APP mice with normal BMAL1 expression (black, AAV-GFP–labeled Cre − ; APP). Dots represent normalized mean hourly ISF Aβ levels in 12-h:12-h L:D conditions (shaded gray) ± SEM ( n = 4–5 mice per genotype), and curves represent cosinor method fits. (F) Bar graph showing the effect of targeted hippocampal Bmal1 deletion on the rhythmicity of ISF Aβ as indicated by cosinor circadian amplitudes. Means were NS by two-tailed t test (P = 0.23). Error bars indicate SEM.

Article Snippet: The following antibodies were used: BMAL1 rabbit polyclonal (1:2,000; 2288; Novus Biologicals) and a monoclonal Aβ 1–13 (1:1,000; HJ3.4) made in-house ( ).

Techniques: Transgenic Assay, In Vivo, Injection, Labeling, Expressing, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: Regulation of amyloid-β dynamics and pathology by the circadian clock

doi: 10.1084/jem.20172347

Figure Lengend Snippet: Bmal1 deletion throughout in the brain but sparing the SCN does not markedly affect Aβ levels or hippocampal amyloid pathology. (A) Representative images showing normal BMAL1 expression in the hippocampus and SCN regions from a control mouse ( Cre − ). (B) Representative raw tracing of core body temperature recordings over 7 d from a Cre − mouse (top). Mean body core temperature recordings over 6 d in 12-h:12-h L:D (bottom right), with a χ 2 periodogram (bottom left) showing a dominant 24-h rhythm present in a control Cre − mouse ( n = 3). (C) Representative images of a Nestin-Cre + ;Bmal1 f/f ( Nes-Bmal1 KO ) mouse showing loss of hippocampal but not SCN BMAL1 expression. OC, optic chiasm. (D) Representative raw tracing of core body temperature recordings over 7 d from a Nes-Bmal1 KO mouse (top). Mean body core temperature recordings over 6 d in 12-h:12-h L:D (bottom right), with a χ 2 periodogram (bottom left) showing a dominant 24-h rhythm present in a Nes-Bmal1 KO mouse ( n = 3). (E) Graph showing hourly hippocampal ISF-soluble Aβ levels in Cre − ;Bmal1 f/f ;APPPS1-21 (black) and Nestin-Cre + ; Bmal1 f/f ;APPPS1-21 ( Nes-Bmal1 KO;APP, orange) mice. Squares represent the normalized mean hourly ISF Aβ level in 12h:12–h L:D conditions (shaded gray) ± SEM ( n = 5 mice per genotype), and curves represent cosinor method fits. (F) Bar graph showing rhythmicity of ISF Aβ as indicated by cosinor circadian amplitudes in Cre − ;APPPS1-21 (black) and Nes-Bmal1 KO;APP (orange) mice. Means were NS by two-tailed t test (P = 0.26). (G) Representative images showing X34 staining of fibrillar Aβ plaques in the hippocampus of 4-mo Cre − ;APPPS1-21 (top) and Nes-Bmal1 KO;APP mice (bottom). The hippocampus is outlined by dashed gray lines. Bars: (A and C, top, and G) 500 µm; (A and C, bottom) 250 µm. (H) Bar graph showing quantification of immunoreactive Aβ (HJ3.4 antibody staining, left) and fibrillar Aβ (X34 staining, right) plaque burden within the hippocampus of Cre − APPPS1-21 (black) and Nes-Bmal1 KO;APP (orange) mice ( n = 9–14 mice per genotype). Bars represent means ± SEM (two-tailed t test; P = 0.88 for the immunoreactive Aβ comparison, and P = 0.064 for the fibrillar Aβ comparison).

Article Snippet: The following antibodies were used: BMAL1 rabbit polyclonal (1:2,000; 2288; Novus Biologicals) and a monoclonal Aβ 1–13 (1:1,000; HJ3.4) made in-house ( ).

Techniques: Expressing, Control, Two Tailed Test, Staining, Comparison

Journal: The Journal of Experimental Medicine

Article Title: Regulation of amyloid-β dynamics and pathology by the circadian clock

doi: 10.1084/jem.20172347

Figure Lengend Snippet: Global Bmal1 deletion disrupts Aβ rhythmicity. (A) Representative images showing decreased BMAL1 expression in the hippocampus and SCN regions from a global Bmal1 KO mouse. (B) Representative raw tracing of core body temperature recordings over 6 d from a Bmal1 KO mouse. Mean body core temperature recordings over 6 d in 12-h:12-h L:D (bottom right), with a χ 2 periodogram (bottom left) showing a loss of the dominant 24-h rhythm in a global Bmal1 KO mouse ( n = 3). (C) Graph showing hippocampal ISF-soluble Aβ levels in WT (labeled control, black) and global Bmal1 KO mice (red). Squares represent the normalized mean murine ISF Aβ level pooled every 3 h in 12h:12–h L:D conditions (shaded gray) ± SEM ( n = 7 mice per genotype), and curves represent cosinor method fits. (D) Bar graph showing rhythmicity of ISF Aβ as indicated by cosinor circadian amplitudes in WT (labeled control, black) and Bmal1 KO (red) mice. *, P = 0.002 by two-tailed t test. (E) Bar graph depicting daily mean hippocampal ISF Aβ levels in WT (black) and Bmal1 KO mice (red). Means were NS by two-tailed t test (P = 0.71). Means + SEM are shown; n = 7 mice/group.

Article Snippet: The following antibodies were used: BMAL1 rabbit polyclonal (1:2,000; 2288; Novus Biologicals) and a monoclonal Aβ 1–13 (1:1,000; HJ3.4) made in-house ( ).

Techniques: Expressing, Labeling, Control, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: Regulation of amyloid-β dynamics and pathology by the circadian clock

doi: 10.1084/jem.20172347

Figure Lengend Snippet: Global Bmal1 deletion accelerates amyloid plaque deposition. (A) Representative images showing normal BMAL1 expression in the hippocampus and SCN regions from a tamoxifen-treated Cre − mouse. (B) Representative raw tracing of core body temperature recordings over 7 d from a Cre − mouse (top). Mean body core temperature recordings over 6 d in 12-h:12-h L:D (bottom right), with a χ 2 periodogram (bottom left) showing a dominant 24-h rhythm present in a Cre- control mouse ( n = 3). (C) Representative images of a tamoxifen-treated CAG-Cre ERT2+ ;Bmal1 f/f ( Bmal1 iKO ) mouse showing a loss of both hippocampal and SCN BMAL1 expression. OC, optic chiasm. (D) Representative raw tracing of core body temperature recordings over 7 d from an inducible Bmal1 KO mouse (top). Mean body core temperature recordings over 6 d in 12-h:12-h L:D are arrhythmic (bottom right), with a χ 2 periodogram (bottom left) showing a loss of the dominant 24-h rhythm ( n = 3). (E) Representative images showing X34 staining of fibrillar Aβ plaques in the hippocampus of 4-mo Cre − ;Bmal1 f/f ;APP ( Cre - ;APP , top) and Bmal1 iKO ; APP (red) mice (bottom). The hippocampus is outlined by dashed gray lines. Bars: (A and C, top, and E) 500 µm; (A and C, bottom) 300 µm. (F) Bar graph showing quantification of immunoreactive Aβ (HJ3.4 antibody staining, left) and fibrillar Aβ (X34 staining, right) plaque burden within the hippocampus of Cre − ;APP (black) and Bmal1 iKO ; APP (red) mice ( n = 8–13 mice per genotype). Bars represent means ± SEM (two-tailed t test; P = 0.001 for the immunoreactive Aβ comparison, and P = 0.001 for the fibrillar Aβ comparison). *, P < 0.05.

Article Snippet: The following antibodies were used: BMAL1 rabbit polyclonal (1:2,000; 2288; Novus Biologicals) and a monoclonal Aβ 1–13 (1:1,000; HJ3.4) made in-house ( ).

Techniques: Expressing, Control, Staining, Two Tailed Test, Comparison

Journal: PLoS ONE

Article Title: Core circadian clock transcription factor BMAL1 regulates mammary epithelial cell growth, differentiation, and milk component synthesis

doi: 10.1371/journal.pone.0248199

Figure Lengend Snippet: ( a) Homer annotation analysis of BMAL1 ChIP-seq peak location shows frequency of distribution across the genome in undifferentiated (UNDIFF) and lactogen differentiated (DIFF) HC11 cultures. 3UTR, 3’ untranslated region; TTS, terminal transcription site; pseudo, pseudogene; 5UTR, 5’ untranslated region. (b) Venn diagram illustrating the overlap and number of unique Ensembl gene IDs of protein coding genes with transcriptional start site nearest to BMAL1 ChIP-seq peaks in the two undifferentiated (UNDIFF1 and UNDIFF2) and two differentiated (DIFF1 and DIFF2) samples.

Article Snippet: For IP analysis of specificity of ChIP grade antibody BMAL1 (Abcam; ab3350), rabbit polyclonal antibody to BMAL1 (Abcam; ab3350) was added to 200 μg protein in 200 μl cold PBST (PBS pH 7.4 with 0.02% Tween-20), and rotated overnight at 4°C.

Techniques: ChIP-sequencing

Journal: PLoS ONE

Article Title: Core circadian clock transcription factor BMAL1 regulates mammary epithelial cell growth, differentiation, and milk component synthesis

doi: 10.1371/journal.pone.0248199

Figure Lengend Snippet: Representative categories enriched with protein coding genes nearest to BMAL1 ChIP-seq overlapping peaks.

Article Snippet: For IP analysis of specificity of ChIP grade antibody BMAL1 (Abcam; ab3350), rabbit polyclonal antibody to BMAL1 (Abcam; ab3350) was added to 200 μg protein in 200 μl cold PBST (PBS pH 7.4 with 0.02% Tween-20), and rotated overnight at 4°C.

Techniques: Clinical Proteomics, Membrane, Activity Assay, Ubiquitin Proteomics, Binding Assay

Journal: PLoS ONE

Article Title: Core circadian clock transcription factor BMAL1 regulates mammary epithelial cell growth, differentiation, and milk component synthesis

doi: 10.1371/journal.pone.0248199

Figure Lengend Snippet: Representative categories enriched with potential BMAL1 target genes identified using ChIP-seq that overlapped with genes that showed circadian rhythms of expression in mammary glands and BMAL1 targets identified in hepatic tissue.

Article Snippet: For IP analysis of specificity of ChIP grade antibody BMAL1 (Abcam; ab3350), rabbit polyclonal antibody to BMAL1 (Abcam; ab3350) was added to 200 μg protein in 200 μl cold PBST (PBS pH 7.4 with 0.02% Tween-20), and rotated overnight at 4°C.

Techniques: Expressing, Ubiquitin Proteomics, Ligand Binding Assay

Journal: PLoS ONE

Article Title: Core circadian clock transcription factor BMAL1 regulates mammary epithelial cell growth, differentiation, and milk component synthesis

doi: 10.1371/journal.pone.0248199

Figure Lengend Snippet: (a)Western blot analysis of BMAL1 protein abundance in HC11 and monoclonal colonies (1A, 1B, 1C, 2A, 2B, 2C, 2D) established from CRISPR-CAS transfected with guide RNA (gRNA) targeting BMAL1 gene post monoclonal selection. BMAL1 is absent in colonies 1C and 2D and decreased in the remaining colonies compared to WT HC11. Monoclonal 1C culture was used in all subsequent experiments, and referred to as BMAL1-KO. Data are representative of two western blots. (b) Temporal analysis of Per2 expression in WT HC11 (solid line) and BMAL1-KO (dashed line) cultures. For this experiment cells were grown to confluence in growth media. Media was changed to lactogen media for 2 hr to synchronize clocks, as we previously have shown this synchronizes clocks in HC11 cells . At completion of 2 hr lactogen treatment (time 0 hr), cells were rinsed with PBS and cultured in growth media for remainder of the experiment. Cells were collected for isolation of total RNA every 4 hr over a 48 hr period beginning at 0 hr. Per2 was measured with RT-qPCR, and levels were expressed relative to mean ΔCT of HC11 across all time points. Cosinor analysis found mesor (-0.12 and -0.68), amplitude (1.58 and 0.70), acrophase (0.78 and -0.56), R 2 (0.85 and 0.47) and p -value (8.05 E-5 and 0.04) of fit to a 24 hr rhythm, respectively, for HC11 and BMAL1-KO lines. Data represent n = 3 wells/line and 2 experimental replicates. (c) BMAL1 protein abundance in UNDIFF and DIFF cultures measured using ELISA. Data are expressed as mean μg of BMAL1/mg protein ± standard deviation of three samples per treatment; * indicates difference at p <0.05. Data represent n = 3 protein isolates/line/state of differentiation.

Article Snippet: For IP analysis of specificity of ChIP grade antibody BMAL1 (Abcam; ab3350), rabbit polyclonal antibody to BMAL1 (Abcam; ab3350) was added to 200 μg protein in 200 μl cold PBST (PBS pH 7.4 with 0.02% Tween-20), and rotated overnight at 4°C.

Techniques: Western Blot, Quantitative Proteomics, CRISPR, Transfection, Selection, Expressing, Cell Culture, Isolation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: PLoS ONE

Article Title: Core circadian clock transcription factor BMAL1 regulates mammary epithelial cell growth, differentiation, and milk component synthesis

doi: 10.1371/journal.pone.0248199

Figure Lengend Snippet: (a) Eight day growth curve analysis was performed by plating 100,000 cells/well on day 0 in 6 well dish of wild type HC11(black line) and BMAL1-KO (gray line), two wells/treatment were collected and counted every two days; values are mean ± standard deviation across five experiments. Two way ANOVA found that line and day significantly affected ( p <0.05) number of cells. Data represent five experimental replicates, with n = 2 wells/line per experiment. (b) MTT assay was performed by plating HC11 (black bars) and BMAL1-KO (gray bars) cells at 10,000 cells/well in a 96-well plate; on days 2, 4, 6 and 8 of culture, MTT assay was performed. ANOVA found line and day significantly affected NADH levels; values are mean ± standard deviation; * indicates difference between lines at p <0.05 across 3 replicate experiments. Data represent three replicate experiments collected from n = 3 wells/line/day. (c) The intensity of MTT staining per cell across three images on each day in HC11 (black) and BMAL1-KO (gray) cultures was quantified. Values are mean intensity per cell ± standard deviation. A significant difference at p <0.05 is indicated by *. (d) Images of cells were captured following staining with the MTT assay on day 2 and 6 of culture. (e) Cells were collected from 100 mm dishes (plating density was 100,000 cells/ml) for fluorescence activated cell sorting (FACS) to determine percent of cells in G1/G0 and S/G2/M phases following labeling with propidium iodide across 8 days of culture. Values are mean across 5 experiments, with ANOVA analysis finding that day had an effect ( p <0.05) on proportion of cells in phases, but there was no difference between HC11 (black) and BMAL1-KO (gray). (f) FACS analysis for dead or dying cells (cells or events with <2N) in HC11 (black) and BMAL1-KO (gray). Values are percent of total events ± standard error; * indicates difference at p <0.05. (g) RT-qPCR analysis of Ccnd1 and Sod3 expression in undifferentiated (UNDIFF) cultures of HC11 (black) and BMAL1-KO (gray) cells. Values are mean across triplicate samples and two experimental replicates, normalized to express fold-change relative to mean of HC11 ± standard deviation using delta-delta cycle threshold method; Student t-test analysis * indicates difference between lines at p <0.05. (h) Reactive oxygen species (ROS) assay of HC11 and BMAL1 cells on day 3 and 4 of culture. Two-way ANOVA found day and line affected ( p <0.05) ROS levels; * indicates difference between lines at p <0.05 across 3 replicate experiments n = 3 wells/line/day; values are mean of arbitrary units (AU) ± standard deviation.

Article Snippet: For IP analysis of specificity of ChIP grade antibody BMAL1 (Abcam; ab3350), rabbit polyclonal antibody to BMAL1 (Abcam; ab3350) was added to 200 μg protein in 200 μl cold PBST (PBS pH 7.4 with 0.02% Tween-20), and rotated overnight at 4°C.

Techniques: Standard Deviation, MTT Assay, Staining, Fluorescence, FACS, Labeling, Quantitative RT-PCR, Expressing, ROS Assay

Journal: PLoS ONE

Article Title: Core circadian clock transcription factor BMAL1 regulates mammary epithelial cell growth, differentiation, and milk component synthesis

doi: 10.1371/journal.pone.0248199

Figure Lengend Snippet: RT-qPCR analysis of (a) Tph1 and (b) Slc6a4 (Sert) in undifferentiated (UNDIFF) and lactogen differentiated (DIFF) HC11 (black) and BMAL1-KO (gray) cultures. Values are mean across triplicate samples and two experimental replicates. To calculate relative difference, data were normalized to mean ΔCT of HC11 and fold change difference was determined using the delta-delta CT method; ANOVA and post-hoc Tukey test analysis indicated with differing letter reflecting difference at p <0.05. (c) ChIP-qPCR analysis of Slc6a4 (Sert) promoter region using primers that targeted two sites that contained E-box sequences beginning at -42 and -1282 nucleotide bases upstream of the transcriptional start site in undifferentiated HC11 cultures; values are mean across four samples, normalized to express fold-change relative to mean CT of mock. A 2-fold difference was considered a positive ChIP.

Article Snippet: For IP analysis of specificity of ChIP grade antibody BMAL1 (Abcam; ab3350), rabbit polyclonal antibody to BMAL1 (Abcam; ab3350) was added to 200 μg protein in 200 μl cold PBST (PBS pH 7.4 with 0.02% Tween-20), and rotated overnight at 4°C.

Techniques: Quantitative RT-PCR, ChIP-qPCR

Journal: PLoS ONE

Article Title: Core circadian clock transcription factor BMAL1 regulates mammary epithelial cell growth, differentiation, and milk component synthesis

doi: 10.1371/journal.pone.0248199

Figure Lengend Snippet: RT-qPCR analysis of (a) Ppara and (b) Csn2 in undifferentiated (UNDIFF) and lactogen differentiated (DIFF) HC11 and BMAL1-KO cultures. Values are mean across triplicate samples and two replicate experiments, normalized to express fold-change relative to mean of HC11 ± standard deviation using delta-delta cycle threshold method. Note the y-axis in Ppara is fold change, whereas y-axis for Csn2 is log base 2 of fold change due to large induction. ANOVA and post-hoc Tukey test analysis findings is indicated by differing letter reflecting difference at p<0.05. (c) ELISA quantification of CSN2 protein in UNDIFF and DIFF HC11 (black) and BMAL1-KO (gray) cultures. Values are mean concentration ± standard deviation across triplicate samples. ANOVA and post-hoc Tukey test analysis findings is indicated by differing letter reflecting difference at p<0.05. (d) RT-qPCR analysis of Prlr in UNDIFF and lactogen DIFF HC11 (black) and BMAL1-KO (gray) cultures. Values are mean across triplicate samples, normalized to express fold-change relative to mean of HC11 ± standard deviation using delta-delta cycle threshold method. Note the y-axis is fold change. ANOVA and post-hoc Tukey test analysis findings is indicated by differing letter reflecting difference at p<0.05. (e) Images of two and a half dimensional drip gel cultures of HC11 and BMAL-KO cells taken with phase-contrast microscopy after 7 days of incubation in lactogen media. Cells were plated at 13,000 cells/well.

Article Snippet: For IP analysis of specificity of ChIP grade antibody BMAL1 (Abcam; ab3350), rabbit polyclonal antibody to BMAL1 (Abcam; ab3350) was added to 200 μg protein in 200 μl cold PBST (PBS pH 7.4 with 0.02% Tween-20), and rotated overnight at 4°C.

Techniques: Quantitative RT-PCR, Standard Deviation, Enzyme-linked Immunosorbent Assay, Concentration Assay, Microscopy, Incubation

Journal: iScience

Article Title: BMAL1 upregulates STX17 levels to promote autophagosome-lysosome fusion in hippocampal neurons to ameliorate Alzheimer's disease

doi: 10.1016/j.isci.2024.111413

Figure Lengend Snippet: Figure 5. The AD model showed circadian rhythm disturbance and decreased Bmal1 expression (A) The protocol of synchronize in vivo and in vitro. (B) APP/PS1 mice displayed circadian rhythm disorders, increased daytime activity, and prolonged free-running cycles.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-LC3B antibody Abcam Cat #ab192890 BMAL1 (D2L7G) Rabbit mAb Cell Signaling Technology Cat #14020S STX17 Polyclonal antibody Proteintech Cat #17815-1-AP VAMP8 Polyclonal antibody Proteintech Cat #15546-1-AP SNAP29 Polyclonal antibody Proteintech Cat #12704-1-AP Anti-SQSTM1 / p62 antibody Abcam Cat #ab109012 Anti-Amyloid Precursor Protein antibody Abcam Cat #ab32136 Bacterial and virus strains Lentivirus-overexpressing STX17 Shanghai Genechem N/A Lentivirus-overexpressing BMAL1 Shanghai Genechem N/A STX17-siRNA Hanbio Biotechnology N/A Chemicals, peptides, and recombinant proteins RNAiso Plus TaKaRa Cat #9109 RIPA buffer Boster Cat #AR0102 Glutaraldehyde,2.5%(EM Grade) Solarbio Cat #P1126 Critical commercial assays PrimeScript RT Master Mix TaKaRa Cat #RR036A SYBR Premix Ex TaqTMII TaKaRa Cat #DRR041A Experimental models: Cell lines HT22 Chinese Academy of Sciences Cell Bank CSTR:19375.09.3101MOUGNM47 Experimental models: Organisms/strains SPF mice Model Animal Research Center APP/PS1 transgenic mice Software and algorithms ImageJ ImageJ https://imagej.nih.gov/ij/ GraphPad Prism GraphPad Prism https://www.graphpad.com/ Other Code of GSE21779 GEO https://www.ncbi.nlm.nih.gov/gds/ Code for data analysis and visualization bioinformatics https://www.bioinformatics.com.cn Code for revealed the binding sites JASPAR http://jaspar.genereg.net/ Data: Western Blot and Microscopy data Lead contact 163.wangxh@163.com

Techniques: Expressing, In Vivo, In Vitro, Activity Assay

Journal: iScience

Article Title: BMAL1 upregulates STX17 levels to promote autophagosome-lysosome fusion in hippocampal neurons to ameliorate Alzheimer's disease

doi: 10.1016/j.isci.2024.111413

Figure Lengend Snippet: Figure 6. BMAL1 regulates STX17 to affect autophagy and amyloid deposition (A) JASPAR analysis revealed the recognition sites of BMAL1 on the promoter sequence of STX17. (B) Detection of luciferase activity after the STX17 promoter sequence plasmid and BMAL1 plasmid were transfected into HT22 cells. (C) Autophagic flow was partially restored in APP-overexpressed HT22 after BMAL1 overexpression. (D) Amyloid deposition decreases after BMAL1 overexpression. n = 6; scale bar, 50 mm. *p < 0.05 vs. the Lv-OE-APP+Lv-NC group; **p < 0.01 vs. the Lv-OE- APP+Lv-NC group. Data are represented as mean ± SD.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-LC3B antibody Abcam Cat #ab192890 BMAL1 (D2L7G) Rabbit mAb Cell Signaling Technology Cat #14020S STX17 Polyclonal antibody Proteintech Cat #17815-1-AP VAMP8 Polyclonal antibody Proteintech Cat #15546-1-AP SNAP29 Polyclonal antibody Proteintech Cat #12704-1-AP Anti-SQSTM1 / p62 antibody Abcam Cat #ab109012 Anti-Amyloid Precursor Protein antibody Abcam Cat #ab32136 Bacterial and virus strains Lentivirus-overexpressing STX17 Shanghai Genechem N/A Lentivirus-overexpressing BMAL1 Shanghai Genechem N/A STX17-siRNA Hanbio Biotechnology N/A Chemicals, peptides, and recombinant proteins RNAiso Plus TaKaRa Cat #9109 RIPA buffer Boster Cat #AR0102 Glutaraldehyde,2.5%(EM Grade) Solarbio Cat #P1126 Critical commercial assays PrimeScript RT Master Mix TaKaRa Cat #RR036A SYBR Premix Ex TaqTMII TaKaRa Cat #DRR041A Experimental models: Cell lines HT22 Chinese Academy of Sciences Cell Bank CSTR:19375.09.3101MOUGNM47 Experimental models: Organisms/strains SPF mice Model Animal Research Center APP/PS1 transgenic mice Software and algorithms ImageJ ImageJ https://imagej.nih.gov/ij/ GraphPad Prism GraphPad Prism https://www.graphpad.com/ Other Code of GSE21779 GEO https://www.ncbi.nlm.nih.gov/gds/ Code for data analysis and visualization bioinformatics https://www.bioinformatics.com.cn Code for revealed the binding sites JASPAR http://jaspar.genereg.net/ Data: Western Blot and Microscopy data Lead contact 163.wangxh@163.com

Techniques: Sequencing, Luciferase, Activity Assay, Plasmid Preparation, Transfection, Over Expression

Journal: iScience

Article Title: PKA-mediated BMAL1 phosphorylation promotes β 1 -adrenoceptor autoantibody-induced cardiomyocyte death

doi: 10.1016/j.isci.2025.112786

Figure Lengend Snippet: β 1 -AA increases Ser42 phosphorylation of BMAL1 in cardiomyocytes (A and C) Expression of Ser42-phosphorylated BMAL1 (pSer42-BMAL1) in myocardial tissues from control and β 1 -AA groups at different time points. ∗ p < 0.05 vs. control; ∗∗ p < 0.01 vs. control; n = 6. (B and D) pSer42-BMAL1 expression in H9c2 cells in the presence or absence of β 1 -AA at different time points. ∗ p < 0.05 vs. control; n = 5. (E) Expression levels of pSer42-BMAL1 in nuclear and cytosolic fractions of H9c2 cells at CT8 were detected by western blot, with GAPDH (cytoplasm) and histone H3 (nucleus) serving as markers. Data are represented as mean ± SEM.

Article Snippet: Phospho-BMAL1 (Ser42) , Cell Signaling Technology , Cat#13936; RRID: AB_2798348.

Techniques: Phospho-proteomics, Expressing, Control, Western Blot

Journal: iScience

Article Title: PKA-mediated BMAL1 phosphorylation promotes β 1 -adrenoceptor autoantibody-induced cardiomyocyte death

doi: 10.1016/j.isci.2025.112786

Figure Lengend Snippet: Ser42 phosphorylation mediates β 1 -AA-induced cytoplasmic accumulation of BMAL1 (A) BMAL1 expression levels in cytoplasmic and nuclear fractions of phosphomimetic (S42D) and dephosphomimetic (S42A) mutant cells were analyzed by western blot. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. (B) mRNA expression levels of Per2 and Nr1d1 in WT, S42D, and S42A groups were measured by real-time PCR. ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001. (C) BMAL1 expression levels in cytoplasmic and nuclear fractions of the S42A mutant cells after β 1 -AA treatment were analyzed by western blot. ∗ p < 0.05; ∗∗ p < 0.01; ns: no significant difference. (D) mRNA expression levels of Per2 and Nr1d1 in the S42A mutant cells after β 1 -AA treatment were measured by real-time PCR. ∗∗ p < 0.01; ∗∗∗ p < 0.001; ns: no significant difference. (E) Cell viability of the S42A mutant cells in the presence or absence of β 1 -AA. ∗∗∗∗ p < 0.0001; ns: no significant differerce. Data are represented as mean ± SEM.

Article Snippet: Phospho-BMAL1 (Ser42) , Cell Signaling Technology , Cat#13936; RRID: AB_2798348.

Techniques: Phospho-proteomics, Expressing, Mutagenesis, Western Blot, Real-time Polymerase Chain Reaction

Journal: iScience

Article Title: PKA-mediated BMAL1 phosphorylation promotes β 1 -adrenoceptor autoantibody-induced cardiomyocyte death

doi: 10.1016/j.isci.2025.112786

Figure Lengend Snippet: β 1 -AA promotes BMAL1 phosphorylation by enhancing PKA activity (A) Immunoprecipitation revealed the interaction between BMAL1 and PKA in cardiomyocytes, and PKA increased BMAL1 phosphorylation in the presence of β 1 -AA. The PKA substrate motif antibody was employed to enrich proteins phosphorylated by PKA, followed by immunoblotting with BMAL1-specific antibodies to verify BMAL1 in the complexes. (B) pSer42-BMAL1 expression levels were detected by western blot in control group, β 1 -AA group, and β 1 -AA+H89 group. ∗∗ p < 0.01. (C) pSer42-BMAL1 and PRKACA expression levels were detected by western blot in si- NC group, si -Prkaca -1 group, and si -Prkaca -2 group in the presence or absence of β 1 -AA. ∗∗∗ p < 0.001 vs. si- NC . (D) Schematic illustration of the proposed mechanism: β 1 -AA enhances BMAL1 phosphorylation at Ser42 by activating PKA, leading to cytoplasmic accumulation of phosphorylated BMAL1, which suppresses the transcription of downstream target genes Per2 and Nr1d1 and promotes cardiomyocyte death. Data are represented as mean ± SEM.

Article Snippet: Phospho-BMAL1 (Ser42) , Cell Signaling Technology , Cat#13936; RRID: AB_2798348.

Techniques: Phospho-proteomics, Activity Assay, Immunoprecipitation, Western Blot, Expressing, Control

Journal: Cell

Article Title: Genomic decoding of neuronal depolarization by stimulus-specific NPAS4 heterodimers

doi: 10.1016/j.cell.2019.09.004

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse monoclonal anti-ARNTL1 (clone B-1) , Santa Cruz Biotechnology , Cat#sc-365645; RRID:AB_10841724.

Techniques: Virus, Recombinant, RNAscope, Multiplex Assay, Staining, RNA Immunoprecipitation, In Situ, Fluorescence, Derivative Assay, Infection, Library Amplification, Plasmid Preparation, Software