Journal: bioRxiv

Article Title: ARIH1 deficiency impairs spatial learning and memory via GIRK2 upregulation in hippocampal CaMKII-expressing neurons in mice

doi: 10.1101/2025.03.10.625121

Figure Lengend Snippet: ( A ) Workflow of quantitative proteomic analysis. Proteomic data were obtained from mice hippocampus, n =3. ( B ) Heatmaps of differentially expressed (DE) proteins in WT and ARIH1 +/- hippocampal samples. ( C ) Volcano plot for DE proteins (139 upregulated, 66 downregulated) in ARIH1 +/- hippocampal samples compared with WT hippocampus. Red and blue dots indicate statistical significance DE proteins. The hippocampal tissue of WT and ARIH1 +/- mice were dissected and whole lysis was prepared for immunoblot analysis ( D ) and total RNA was prepared for qPCR analysis ( E , F ) as described in the methods. The qPCR was probed by mouse ARIH1 or GIRK2 primers, n =4. The immunoblot was probed by anti-ARIH1 or anti-GIRK2 antibody. The mean intensity of bands was quantified using Image J and normalized to corresponding loading controls, n =4. * p < 0.05, ** p < 0.01, *** p < 0.001, ns . not significant, unpaired Student’s t test ( D , E , F ). Mean ± SEM.

Article Snippet: The following antibodies were used: anti-ARIH1 (Ab3891, Abcam), anti-ARIH1 (NBP2-57888, Novus Biologicals), anti-GIRK2 (21647-1-AP, Proteintech), anti- GAPDH (60004-1-lg, Proteintech), anti-β-actin (66009-1-Ig, Proteintech), anti-Flag (20543-1-AP, Proteintech), anti-HA (51064-2-AP, Proteintech), anti-CaMKII (ab52476, Abcam), anti-GFAP (ab7260, Abcam), anti-Ib1 (011-27991, Wako), anti-Parvalbumin (ab32895, Abcam), and anti-Somatostatin (ab140665, Abcam).

Techniques: Lysis, Western Blot

Journal: bioRxiv

Article Title: ARIH1 deficiency impairs spatial learning and memory via GIRK2 upregulation in hippocampal CaMKII-expressing neurons in mice

doi: 10.1101/2025.03.10.625121

Figure Lengend Snippet: HT-22 cells were infected with lentivirus expressing scramble (NC) or Arih1-shRNA of two different on-target sequence (948, 949). NC and Arih1 knockdown cells were selected and maintained in cell culture media supplemented with puromycin (2.5μg/ml for selection, and 1μg/ml for maintaining) as described in the methods. ( A ) Whole cell lysates were prepared from HT-22 NC and Arih1 knockdown cells for immunoblot analysis probed by anti-Arih1 or anti-Girk2 antibodies. ( B ) Arih1 knockdown cells were transfected with vector or Flag-Arih1 plasmids. After 48hours transfection, whole cell lysates were prepared for immunoblot analysis probed by anti-Girk2, anti-Arih1, or anti-Flag antibodies. ( C ) HT-22 NC and Arih1 knockdown cells were transfected with Flag-Ub and HA-Girk2 plasmids. After 48hours transfection, whole cell lysates were prepared and immunoprecipitation was performed to examine the ubiquitination of Girk2 using anti-Flag or anti-HA antibodies. Showing blots are representative of at least 3 independent experiments. The mean intensity of bands was quantified using Image J and normalized to corresponding loading controls ( A 1 -A 2 , B 1 -B 4 ), or to corresponding input loadings ( C 1 ). ( D , E ) The total RNA was prepared from HT-22 NC and Arih1 knockdown cells for qPCR analysis with mouse ARIH1 or GIRK2 primers, n =5. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns . not significant, one-way ANOVA followed by Tukey’s test, n =3-6. Mean ± SEM.

Article Snippet: The following antibodies were used: anti-ARIH1 (Ab3891, Abcam), anti-ARIH1 (NBP2-57888, Novus Biologicals), anti-GIRK2 (21647-1-AP, Proteintech), anti- GAPDH (60004-1-lg, Proteintech), anti-β-actin (66009-1-Ig, Proteintech), anti-Flag (20543-1-AP, Proteintech), anti-HA (51064-2-AP, Proteintech), anti-CaMKII (ab52476, Abcam), anti-GFAP (ab7260, Abcam), anti-Ib1 (011-27991, Wako), anti-Parvalbumin (ab32895, Abcam), and anti-Somatostatin (ab140665, Abcam).

Techniques: Infection, Expressing, shRNA, Sequencing, Knockdown, Cell Culture, Selection, Western Blot, Transfection, Plasmid Preparation, Immunoprecipitation

Journal: bioRxiv

Article Title: ARIH1 deficiency impairs spatial learning and memory via GIRK2 upregulation in hippocampal CaMKII-expressing neurons in mice

doi: 10.1101/2025.03.10.625121

Figure Lengend Snippet: Schematic of the AAV-ARIH1 shRNA ( A ) and brain injection site ( B ) in mice. ( C ) The MWM experiment is composed of 3 sections which are adaptation (day 1), training (day 2 to 7), and testing (day 8). The latency on day 1 as well as the average latency (4 quadrants) on day 2 to 7 of AAV-Scramble or AAV-ARIH1 shRNA injected mice were calculated as described in the methods. During test on day 8, the latency ( D ) as well as times crossing the platform area ( E ) of AAV-Scramble or AAV-ARIH1 shRNA injected mice were recorded as described in the methods, n =10-12. ( F ) Representative image for showing the site of injection. Scale bar, 100μm. ( G ) The dorsal hippocampal tissue of AAV-Scr and AAV-ARIH1 shRNA injected mice were dissected and whole cell lysates were prepared for immunoblot analysis probed by anti-ARIH1 antibody. The mean intensity of bands was quantified using Image J and normalized to corresponding loading controls, n =3. ( H ) Representative image for showing co-expression of AAV-GFP with CaMKII, but not with GFAP or IB1. Scale bar, 20μm. ( I ) Representative image of immunostaining of Girk2 in mice with dorsal hippocampal injection of AAV-Scramble or AAV-ARIH1 shRNA. Scale bar, 100μm. ( J ) Quantification of the intensity of Girk2 staining, n =5. ( K ) The latency on day 1 as well as the average latency (4 quadrants) on day 2 to 7 of AAV-ARIH1 shRNA injected mice, administrated with Saline or Tipepidine ( i.p. , 20mg/kg), were calculated as described in the methods. During test on day 8, the latency ( L ) as well as times crossing the platform area ( M ) of AAV-ARIH1 shRNA injected mice, administrated with Saline or Tipepidine, were recorded as described in the methods, n =10-11. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, unpaired Student’s t test ( D , E , G , J , L , M ). * p < 0.05, two-way ANOVA followed by Bonferroni’s test ( C , K ). Mean ± SEM.

Article Snippet: The following antibodies were used: anti-ARIH1 (Ab3891, Abcam), anti-ARIH1 (NBP2-57888, Novus Biologicals), anti-GIRK2 (21647-1-AP, Proteintech), anti- GAPDH (60004-1-lg, Proteintech), anti-β-actin (66009-1-Ig, Proteintech), anti-Flag (20543-1-AP, Proteintech), anti-HA (51064-2-AP, Proteintech), anti-CaMKII (ab52476, Abcam), anti-GFAP (ab7260, Abcam), anti-Ib1 (011-27991, Wako), anti-Parvalbumin (ab32895, Abcam), and anti-Somatostatin (ab140665, Abcam).

Techniques: shRNA, Injection, Western Blot, Expressing, Immunostaining, Staining, Saline

Journal: The New phytologist

Article Title: Receptor-like kinases BIR1 and BIR3 modulate antiviral resistance by different mechanisms.

doi: 10.1111/nph.70216

Figure Lengend Snippet: Fig. 2 Genetic dissection of the antiviral response in bir1-1 Arabidopsis mutants. (a, b) Reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis of tobacco rattle virus (TRV) genomic RNA levels in TRV-infected Arabidopsis wild-type (WT; Columbia-0 (Col-0)) and various mutant combinations, including bir1-1 sobir1-1, sobir1-13, bir2-1, bak1-4 pad4-1 sobir7-1 (a), and bir1-1 sobir1-1 pad4-1, bir1-1 pad4-1 sobir7-1 (b), at 5 d postinoculation (dpi). Morphological phenotypes of each genotype at 5 dpi with TRV are also shown. A noninoculated bir1-1 pad4-1 mutant is included for size comparison. Relative TRV RNA levels were normalized to the CBP20 internal control and compared with WT plants (set to 1). Data are mean SD analyzed by one-way ANOVA followed by Tukey’s multiple comparison test; ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05. Experiments were repeated with similar results. BIR, BAK1-INTERACTING RECEPTOR-LIKE KINASE. BAK1, BRASSINOSTEROID INSENSITIVE1-ASSOCIATED RECEPTOR KINASE 1, PAD4, PHYTOALEXIN DEFICIENT 4. SOBIR, SUPPRESSOR OF BIR1-1.

Article Snippet: Immunoprecipitation of mCherry-tagged BIR1 protein from Arabidopsis leaves was performed using RFP-Trap (Chromotek, Planegg, Germany) according to the manufacturer’s protocol.

Techniques: Dissection, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Virus, Infection, Mutagenesis, Comparison, Control

Journal: The New phytologist

Article Title: Receptor-like kinases BIR1 and BIR3 modulate antiviral resistance by different mechanisms.

doi: 10.1111/nph.70216

Figure Lengend Snippet: Fig. 4 Transcriptional changes associated with BRASSINOSTEROID INSENSITIVE1-ASSOCIATED RECEPTOR KINASE 1 (BAK1)-INTERACTING RECEPTOR-LIKE KINASE1 (BIR1) induction in Arabidopsis. (a) Morphology of representative dexamethasone (DEX)-inducible BIR1-mCherry transgenic Arabidopsis plants (line 9, L9) after 4 d of 30 lM of DEX or mock treatment (H2O). (b) Western blot analysis using anti-mCherry antibody to detect BIR1- mCherry protein accumulation after 4 d of DEX treatment. BIR1 transcript levels in wild-type (WT) and BIR1 L9 plants were quantified using RNA-Seq (Fragments Per Kilobase of transcript sequence per Millions of base-pairs sequenced (FPKM)) and reverse transcription quantitative polymerase chain reaction (RT-qPCR). RT-qPCR values were normalized to the CBP20 internal control and compared with mock-treated (set to 1). Data are mean SD analyzed by unpaired t-test. ***, P < 0.001. (c) Principal component analysis (PCA) of RNA-Seq data from mock-treated WT (Mock-WT), mock-treated BIR1 L9 (Mock-L9), DEX-treated WT (DEX-WT), and DEX-treated BIR1 L9 (DEX-L9) plants. (d) Number of up- or downregulated transcripts (|log2(fold change)| ≥1 and adjusted P-value ≤0.01) in each pairwise comparison. (e) Scatterplot of selected Kyoto Encyclopedia of Genes and Genomes (KEGG) terms of differentially expressed genes (DEGs) between DEX-treated and mock-treated BIR1 L9 plants. Dot size represents the gene count, and color indicates the significance of term enrichment. Gene Ontology (GO) terms with adjusted P-value < 0.05 are significantly enriched.

Article Snippet: Immunoprecipitation of mCherry-tagged BIR1 protein from Arabidopsis leaves was performed using RFP-Trap (Chromotek, Planegg, Germany) according to the manufacturer’s protocol.

Techniques: Transgenic Assay, Western Blot, RNA Sequencing, Sequencing, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Control, Comparison

Journal: The New phytologist

Article Title: Receptor-like kinases BIR1 and BIR3 modulate antiviral resistance by different mechanisms.

doi: 10.1111/nph.70216

Figure Lengend Snippet: Fig. 6 Transient BRASSINOSTEROID INSENSITIVE1-ASSOCIATED RECEPTOR KINASE 1 (BAK1)-INTERACTING RECEPTOR-LIKE KINASE1 (BIR1) expression partially inhibits poly(I:C)-triggered callose deposition at plasmodesmata (PD) in Nicotiana benthamiana. (a) Callose deposition at PDs was visualized by aniline blue staining of epidermal cells in N. benthamiana leaves transiently expressing a 35S-driven BIR1-HA construct or an empty vector (EV). Callose deposition was observed after treatment with H2O (control) or 50 ng ll1 poly(I:C). Bars, 10 lm. (b) Quantification of PD callose content in leaves infiltrated with EV or BIR1-HA in response to H2O, poly(I:C), or flg22 (10 lM). Each dot represents the mean callose intensity at individual PDs (> 100) measured in three to four leaf disks from at least three independent biological replicates per treatment and analyzed by one-way ANOVA followed by Tukey’s multiple comparison test; ****, adjusted P-value < 0.0001; ***, P < 0.001. (c) Western blot analysis using anti-HA and anti-mCherry antibodies to detect BIR1-HA and BIR1-mCherry proteins, respectively, in agroinfiltrated areas at 2 d postinoculation (dpi). (d) Western blot analysis using anti-HA antibodies to detect HA-tagged BIR1 and GFP proteins in areas co-infiltrated with TRV-GFP and EV () or BIR1-HA (+) at 3 and 5 dpi. Red Ponceau is shown as a loading control. (e) Transient expression of BIR1 promotes viral spreading in N. benthamiana leaves. Whole leaves were first infiltrated with Agrobacterium containing BIR1-HA under the 35S promoter, BIR1-mCherry under a dexamethasone (DEX)-inducible promoter, or the corresponding EV, and then spot-infiltrated (indicated by dash circles) into the same area with Agrobacterium carrying TRV-GFP. For DEX-inducible BIR1- mCherry expression, 30 lM DEX was added to the infiltration solution. Photographs were taken under ultraviolet light 2 dpi to visualize GFP fluorescence. Bars, 1 cm. All experiments were repeated with similar results. TRV, tobacco rattle virus; GFP, green fluorescent protein.

Article Snippet: Immunoprecipitation of mCherry-tagged BIR1 protein from Arabidopsis leaves was performed using RFP-Trap (Chromotek, Planegg, Germany) according to the manufacturer’s protocol.

Techniques: Expressing, Staining, Construct, Plasmid Preparation, Control, Comparison, Western Blot, Virus

Journal: The New phytologist

Article Title: Receptor-like kinases BIR1 and BIR3 modulate antiviral resistance by different mechanisms.

doi: 10.1111/nph.70216

Figure Lengend Snippet: Fig. 8 Genetic dissection of the antiviral response in bir3-2 Arabidopsis mutants. (a) Reverse transcription quantitative polymerase chain reaction (RT- qPCR) analysis of tobacco rattle virus (TRV) genomic RNA levels in TRV-infected Arabidopsis wild-type (WT, Columbia-0 (Col-0)) and various mutant combinations, including bir3-2, csa1-2, bir3-2 bak1-4 csa1-2, and bir3-2 bak1-4 eds1-12, at 5 d postinoculation (dpi). (b) RT-qPCR analysis of TRV genomic RNA levels in TRV-infected WT (Col-0), bak1-4, pad4-1, eds1-2, and pad4-1 eds1-2 at 5 dpi. Morphological phenotypes of each genotype at 5 dpi with TRV are also shown. A noninoculated bir3-2 bak1-4 mutant is included for size comparison. Relative TRV RNA levels were normalized to the CBP20 internal control and compared with WT plants (set to 1). Data are mean SD analyzed by one-way ANOVA followed by Tukey’s multiple comparison test; ****, P < 0.0001; **, P < 0.01; *, P < 0.05. Experiments were repeated with similar results. (c) Model for BAK1-INTERACTING RECEPTOR-LIKE KINASE (BIR)-mediated regulation of antiviral defense. The BIR1 and BIR3 proteins are induced during virus infection, influenced by antagonistic interactions between salicylic acid (SA) and jasmonic acid (JA) hormone signaling. Induction of BIR2 during infection is affected by SA, but not JA. BIR1 negatively regulates antiviral defense through mechanisms that may include pattern-triggered immunity (PTI) gene expression and plasmodesmata (PD) callose deposition as well as yet unidentified pathways independent of reactive oxygen species (ROS) or the BAK1, SOBIR1, or PAD4 signaling components. BIR3 represses an antiviral response that relies on the activation of BAK1- and EDS1/PAD4-dependent effector-triggered immunity (ETI), likely involving intracellular nucleotide-binding leucine-rich repeat (NLR) receptors, leading to asymptomatic resistance. Solid arrows indicate activation, blunt-ended arrows denote repression, and dashed arrows represent potential effects on antiviral defenses. BAK1, BRASSINOSTEROID INSENSITIVE1-ASSOCIATED RECEPTOR KINASE 1; CSA1, CONSTITUTIVE SHADE AVOIDANCE 1; EDS1, ENHANCED DISEASE SUSCEPTIBILITY 1; PAD4, PHYTOALEXIN DEFICIENT 4.

Article Snippet: Immunoprecipitation of mCherry-tagged BIR1 protein from Arabidopsis leaves was performed using RFP-Trap (Chromotek, Planegg, Germany) according to the manufacturer’s protocol.

Techniques: Dissection, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Virus, Infection, Mutagenesis, Comparison, Control, Gene Expression, Activation Assay, Binding Assay