phospho ryanodine receptor type 2  (Proteintech)

Journal: bioRxiv

Article Title: FAM84B facilitates tau propagation via RYR3-mediated exocytosis in response to neuroinflammation

doi: 10.64898/2025.12.28.696549

Figure Lengend Snippet: (A) Differentially expressed genes in HEK293 cells in response to FAM84B KO (n=3 each). Genes with p < 0.05 and >2-fold expression change compared to control are marked with red dots. (B) Differentially expressed genes in HEK293 cells in response to FAM84B OE (n=3 each). Genes with p < 0.05 and >2-fold expression change compared to control are marked with red dots. (C) Differentially expressed genes in tau-expressing SH-SY5Y cells in response to FAM84B OE (n=3 each). Genes with p < 0.05 and >1.7-fold expression change compared to cells expressing tau alone are marked with red dots. (D) Venn diagram showing the number of shared genes between RNA-seq datasets in (A–C), with overlapping genes represented in distinct colors (red, green, black, blue). Only the names of membrane-related genes among the shared genes are noted. (E) GO analysis showing cellular compartments of genes identified in (D). The GO terms based on the number of genes are shown. (F) Levels of RYR3 and actin in iPSC-derived CN from HC and patients with AD (n=3 each). (G) Levels of RYR3 and actin in SH-SY5Y cells expressing tau alone or tau with FAM84B-Myc (n=3 each). (H) Colocalization (arrow) of FAM84B and calcium in SH-SY5Y cells expressing tau with mCherry control (C1), and in cells expressing tau with mCherry-FAM84B treated with either DMSO or dantrolene (n=4 each). (I) PHF-1 spread from SH-SY5Y cells expressing Tau-p2a-eGFP with FAM84B to neighboring cells in response to treatment with either DMSO or dantrolene (n=4 each). Arrows indicate recipient cells (red) to which tau has been transmitted. Solid boxes in the images indicate magnified areas. Results are presented as mean ± SEM. Co-localization analysis. (H) Pearson correlation coefficient. Statistical analyses: (A-C, F-I) unpaired two-tailed t -test with Welch’s correction; ns: not significant, *p < 0.05, **p < 0.01, ***p < 0.001. Abbreviations: KO, knockout; OE, overexpression; GO, gene ontology; CN, cortical neuron; HC, healthy control; AD, Alzheimer’s disease; PHF-1, phosphorylated tau at residues S396/S404; FOV, field of view.

Article Snippet: Following blocking, membranes were incubated overnight at 4°C with primary antibodies (1:1,000 dilution), including: PHF-1 (Peter Davies Lab), Tau5 (Gail VW Johnson Lab), Tau (Dako, Agilent Technologies, Inc., 0024), Myc-tag (Cell Signaling Technology, 2276), FAM84B (OriGene, TA501992; Proteintech Group, Inc., Rosemont, IL, USA, 18421-1-AP), Alix (Cell Signaling Technology, 2171), Caveolin-1 (Cell Signaling Technology, 3267), RYR3 (Alomone Labs, Jerusalem, Israel, ARR003), Clathrin-heavy chain (CHC, Cell Signaling Technology, 4796), EEA1 (Cell Signaling Technology, 3288), Rab5 (Cell Signaling Technology, 3547), Rab7 (Cell Signaling Technology, 9367), Rab9 (Cell Signaling Technology, 5118), Rab11 (Cell Signaling Technology, 5589), pSTAT3 (Y705) (Cell Signaling Technology, 9145), STAT3 (Cell Signaling Technology, 9139), p62 (Cell Signaling Technology, 8025), LC3 (Cell Signaling Technology, 3868), HSP70 (Cell Signaling Technology, 4876), LAMP-1 (BD Biosciences, Franklin Lakes, NJ, USA, 611042), LAMP-2 (Sigma-Aldrich, L0668), and beta-actin (Merck Millipore, MAB1501).

Techniques: Expressing, Control, RNA Sequencing, Membrane, Derivative Assay, Two Tailed Test, Knock-Out, Over Expression

Journal: bioRxiv

Article Title: FAM84B facilitates tau propagation via RYR3-mediated exocytosis in response to neuroinflammation

doi: 10.64898/2025.12.28.696549

Figure Lengend Snippet: (A) Venn diagram showing the seven shared transcriptional cofactors between the ENCODE ChIP-seq and CHEA ChIP-seq datasets. The genes are categorized into functional terms and noted. (B) Genomic information of the FAM84B gene obtained from the UCSC Genome Browser. Transcription cofactor clusters identified from the ENCODE ChIP-seq data, with those likely to bind to the regulatory elements of FAM84B, as identified by FANTOM5, highlighted. (C) Levels of phosphorylated STAT3 (Y705) in the cerebral cortex of HC (n=3) and patients with VD (n=3), CBD (n=3), PSP (n=4), PART (n=3), and AD (n=6). (D) Levels of phosphorylated STAT3 (Y705) and total STAT3 in iPSC-derived CN from HC and patients with AD (n=3 each). (E) Levels of phosphorylated STAT3 (Y705), total STAT3, FAM84B, and actin in SH-SY5Y cells treated with either control vehicle or IL-6 (n=3 each). (F) Changes in the levels of phosphorylated STAT3 (Y705), total STAT3, FAM84B, and actin in SH-SY5Y cells expressing STAT3-WT or STAT3-DN in response to treatment with either control vehicle or IL-6 (n=3 each). (G) PHF-1 spread from Tau-p2a-eGFP expressing SH-SY5Y cells to neighboring cells in response to treatment with either control vector or IL-6 (n=4 each). Arrows indicate recipient cells (red) to which tau has been transmitted. Solid boxes in the images indicate magnified areas. (H) PHF-1 spread from IL-6 treated SH-SY5Y cells expressing Tau-p2a-eGFP to neighboring cells in response to treatment with either DMSO or dantrolene (n=4 each). Arrows indicate recipient cells (red) to which tau has been transmitted. Solid boxes in the images indicate magnified areas. (I) Changes in the levels of PHF-1, total tau, phosphorylated STAT3 (Y705), total STAT3, FAM84B (FAM84B-Myc), RYR3, and actin in iPSC-derived CN from HC after infection with AAV and subsequent treatments (n=3 each). (J) Changes in the levels of phosphorylated tau (T181 and S396) in the CM of iPSC-derived CN from HC after infection with AAV and subsequent treatments (n=8 each). In (I–J), the experimental groups are: (1) CN infected with AAV containing tau, (2) CN infected with AAV containing tau and FAM84B-Myc, (3) CN infected with AAV containing tau and treated with IL-6 and TNF-α, (4) CN infected with AAV containing tau and FAM84B-Myc and treated with dantrolene, and (5) CN infected with AAV containing tau, treated with IL-6 and TNF-α, followed by treatment with dantrolene. Results are presented as mean ± SEM. Statistical analyses: (C) ordinary two-way ANOVA; (D-J) unpaired two-tailed t -test with Welch’s correction; ns: not significant, *p < 0.05, **p < 0.01, and ***p < 0.001. Abbreviations: HC, healthy control; VD, vascular dementia; CBD, corticobasal degeneration; PSP, progressive supranuclear palsy; PART, primary age-related tauopathy; AD, Alzheimer’s disease; CN, cortical neuron; WT, wild type; DN, dominant negative; PHF-1, phosphorylated tau at S396/S404 residue; FOV, field of view; AAV, adeno-associated virus; CM, conditioned media.

Article Snippet: Following blocking, membranes were incubated overnight at 4°C with primary antibodies (1:1,000 dilution), including: PHF-1 (Peter Davies Lab), Tau5 (Gail VW Johnson Lab), Tau (Dako, Agilent Technologies, Inc., 0024), Myc-tag (Cell Signaling Technology, 2276), FAM84B (OriGene, TA501992; Proteintech Group, Inc., Rosemont, IL, USA, 18421-1-AP), Alix (Cell Signaling Technology, 2171), Caveolin-1 (Cell Signaling Technology, 3267), RYR3 (Alomone Labs, Jerusalem, Israel, ARR003), Clathrin-heavy chain (CHC, Cell Signaling Technology, 4796), EEA1 (Cell Signaling Technology, 3288), Rab5 (Cell Signaling Technology, 3547), Rab7 (Cell Signaling Technology, 9367), Rab9 (Cell Signaling Technology, 5118), Rab11 (Cell Signaling Technology, 5589), pSTAT3 (Y705) (Cell Signaling Technology, 9145), STAT3 (Cell Signaling Technology, 9139), p62 (Cell Signaling Technology, 8025), LC3 (Cell Signaling Technology, 3868), HSP70 (Cell Signaling Technology, 4876), LAMP-1 (BD Biosciences, Franklin Lakes, NJ, USA, 611042), LAMP-2 (Sigma-Aldrich, L0668), and beta-actin (Merck Millipore, MAB1501).

Techniques: ChIP-sequencing, Functional Assay, Derivative Assay, Control, Expressing, Plasmid Preparation, Infection, Two Tailed Test, Dominant Negative Mutation, Residue, Virus