Journal: Nature Communications

Article Title: A clade of receptor-like cytoplasmic kinases and 14-3-3 proteins coordinate inositol hexaphosphate accumulation

doi: 10.1038/s41467-024-49102-6

Figure Lengend Snippet: a BiFC assay showing the interaction between IPCK1/GRF4 and PI-PLC2 in protoplast. Bar = 10 μm. b Split-LUC assay showing the interaction between PI-PLC2 and IPCK1/GRF4. Cps means the fluorescence value. c Split-LUC assay showing the effect of IPCK1 on the interaction between PI-PLC2 and GRF4 or mutated GRF4. Values are given as mean ± SD from three biological replicates. Data were analyzed by unpaired t test ( **P < 0.01). d – f Yeast three-hybrid assay showing that IPCK1 recruits PI-PLC2 and IPK1/IPK2s to form a potential complex. g Detection of IPK1 protein abundance on the plasma membrane (PM) under WT and T-4m backgrounds. Actin and BRI1 serve as internal references for total protein and plasma membrane protein, respectively. WT was used as a negative control. The number in the panel represents the relative proportion of abundance of PM-associated IPK1 proteins to IPK1 total proteins, which are corrected by internal references, respectively. All experiments were repeated at least three times with similar results.

Article Snippet: For western blotting, the anti-flag antibody (Abcam, 1:10000) was used to detect IPK1 protein, and the anti-actin (1:5000) and BRI1 antibody (1:500) were used as internal reference for total proteins and membrane proteins, respectively.

Techniques: Bimolecular Fluorescence Complementation Assay, Fluorescence, Hybrid Assay, Membrane, Negative Control

Journal: International Journal of Molecular Sciences

Article Title: Cold Acclimation and Deacclimation of Winter Oilseed Rape, with Special Attention Being Paid to the Role of Brassinosteroids

doi: 10.3390/ijms25116010

Figure Lengend Snippet: The accumulation of the putative BRI1 protein and relative gene expression ( COR14 , SERK1 , SERK2 ) in the leaves of the non-acclimated (NA), cold-acclimated (CA), and deacclimated (DA) oilseed rape plant cultivars Pantheon and President. ( A ) The representative original membrane, where the visualised bands correspond to the level of putative BRI1 protein; 15 µg of protein was loaded onto the gel. ( B ) The accumulation of the putative BRI1 protein. ( C ) Relative expression of COR14 . ( D ) Relative expression of SERK1 . ( E ) Relative expression of SERK2 . The transcript levels were calculated relative to actin (endogenous reference gene). Mean values ± SE that are indicated by the same letters did not differ according to Duncan’s test ( p < 0.05). Lowercase letters—comparisons between the NA, CA, and DA plants within each cultivar. Capital letters—comparisons between the NA, CA, and DA plants of both cultivars.

Article Snippet: Next, the membranes were washed four times for five minutes with a TBS-T buffer at RT with agitation, and then they were incubated in the primary antibody (dilution: 1:2000; Anti-BRI1 (AS12 1859), Agrisera, Sweden) at 4 °C overnight.

Techniques: Expressing, Membrane

Journal: Nature Communications

Article Title: Copine proteins are required for brassinosteroid signaling in maize and Arabidopsis

doi: 10.1038/s41467-024-46289-6

Figure Lengend Snippet: a Yeast two-hybrid (Y2H) assay assessing the interaction of the A domain of BON1 with the kinase domain (KD) of SERK4, SERK1 and SERK2. The constructs were co-transformed into yeast cells and grown on selective dropout medium as indicated. b Co-IP assay of BON1-HA and SERK4-FLAG in Arabidopsis protoplasts transfected with the respective encoding constructs. BON1-HA and SERK4-FLAG proteins were detected using anti-HA and anti-FLAG antibodies, respectively. PIN1-HA was used as a negative control. c Split luciferase complementation (SLC) assay showing the interaction between BON1 and SERK4. PIN1 was used as a negative control. d Y2H assays showing the interaction between ZmBON1 and the ZmSERK4-KD. e SLC assay showing the interaction between ZmBON1 and ZmSERK4 in N. benthamiana leaves. ZmCDPK7 was used as a negative control. f SLC assay showing the interaction between BON1 and BRI1 in N. benthamiana leaves. PIN1 was used as a negative control. g Co-IP assay of BON1-HA and BRI1-GFP in Arabidopsis protoplasts transfected with the respective encoding constructs. BON1-HA and BRI1-GFP proteins were detected using anti-HA and anti-GFP antibodies, respectively. PIN1-HA was used as a negative control. All experiments were repeated 3 times with similar results.

Article Snippet: To examine the phosphorylation levels of native BRI1 and SERK3 in pad4-1 and bon1-1 bon2-2 bon3-3 pad4-1 after mock and 2 μM eBL treatment, total proteins were extracted from at least 50 seedling per treatment per genotype as described above and incubated with IgG Magnetic Beads (ROCKLAND, RLK00 1800) coupled with anti-BRI1 (Agrisera, AS12 1859) and anti-SERK3 (Agrisera, AS12 1858) antibodies or with anti-FLAG M2 Magnetic Beads (Sigma, M8823) for 4 h at 4 °C to respectively immunoprecipitate BRI1, SERK3, or SERK4-FLAG.

Techniques: Y2H Assay, Construct, Transformation Assay, Co-Immunoprecipitation Assay, Transfection, Negative Control, Luciferase

Journal: Nature Communications

Article Title: Copine proteins are required for brassinosteroid signaling in maize and Arabidopsis

doi: 10.1038/s41467-024-46289-6

Figure Lengend Snippet: a , c Co-IP assay of BRI1 and SERK4 ( a ) and of BRI1 and SERK3 ( c ) in the pad4-1 and bon1-1 bon2-2 bon3-3 pad4-1 ( bon123pad4-1 ) mutants without or with eBL treatment. IP was performed with FLAG beads ( a ) or α-SERK3 ( c ). Immunoprecipitated SERK4-FLAG ( a ), SERK3 ( c ) and co-immunoprecipitated BRI1 were detected with specific α-FLAG, α-SERK3, and α-BRI1 antibodies, respectively. b , d Quantification of BRI1 associated with SERK4-FLAG ( b ) or SERK3 ( d ) shown in ( a ) and ( c ), respectively. Significant differences were determined by two-tailed Student’s t -tests, asterisks indicate statistically significant differences (* P < 0.05, n = 3 biologically independent samples, ±SD). Black dots represent individual data. e Table showing the phosphopeptide sequences and the phosphorylation sites of the BR signaling components BRI1, SERK1/2/3, BKI1, BSK1, BSK5/8, BSL2, and BSL3. Phosphorylated amino acids are shown in red. f – i Phosphorylation levels of BRI1 ( f , g ), SERK3 ( h ) and SERK4 ( i ) in the pad4-1 and bon1-1 bon2-2 bon3-3 pad4-1 ( bon123pad4-1 ) mutants without or with eBL treatment. Phosphorylated proteins were detected with anti-pSer and anti-pThr antibodies. All experiments were repeated 3 times with similar results.

Article Snippet: To examine the phosphorylation levels of native BRI1 and SERK3 in pad4-1 and bon1-1 bon2-2 bon3-3 pad4-1 after mock and 2 μM eBL treatment, total proteins were extracted from at least 50 seedling per treatment per genotype as described above and incubated with IgG Magnetic Beads (ROCKLAND, RLK00 1800) coupled with anti-BRI1 (Agrisera, AS12 1859) and anti-SERK3 (Agrisera, AS12 1858) antibodies or with anti-FLAG M2 Magnetic Beads (Sigma, M8823) for 4 h at 4 °C to respectively immunoprecipitate BRI1, SERK3, or SERK4-FLAG.

Techniques: Co-Immunoprecipitation Assay, Immunoprecipitation, Two Tailed Test

Journal: Nature Communications

Article Title: Copine proteins are required for brassinosteroid signaling in maize and Arabidopsis

doi: 10.1038/s41467-024-46289-6

Figure Lengend Snippet: In the wild type, BONs interact with SERKs, which are spatially separated from BRI1. Upon BR perception, SERKs move close to BRI1 with the assistance of BONs to form the BRI1-SERKs complex. Then, BRI1 is fully activated by reciprocal phosphorylation of BRI1 and SERKs, and the BR signal is amplified to induce an efficient BR response. In the bon mutants, the interactions between BRI1 and SERKs were affected, resulting in attenuated reciprocal phosphorylation between BRI1 and SERKs, leading to an attenuated BR response.

Article Snippet: To examine the phosphorylation levels of native BRI1 and SERK3 in pad4-1 and bon1-1 bon2-2 bon3-3 pad4-1 after mock and 2 μM eBL treatment, total proteins were extracted from at least 50 seedling per treatment per genotype as described above and incubated with IgG Magnetic Beads (ROCKLAND, RLK00 1800) coupled with anti-BRI1 (Agrisera, AS12 1859) and anti-SERK3 (Agrisera, AS12 1858) antibodies or with anti-FLAG M2 Magnetic Beads (Sigma, M8823) for 4 h at 4 °C to respectively immunoprecipitate BRI1, SERK3, or SERK4-FLAG.

Techniques: Amplification

Journal: Nature Communications

Article Title: Copine proteins are required for brassinosteroid signaling in maize and Arabidopsis

doi: 10.1038/s41467-024-46289-6

Figure Lengend Snippet: a Yeast two-hybrid (Y2H) assay assessing the interaction of the A domain of BON1 with the kinase domain (KD) of SERK4, SERK1 and SERK2. The constructs were co-transformed into yeast cells and grown on selective dropout medium as indicated. b Co-IP assay of BON1-HA and SERK4-FLAG in Arabidopsis protoplasts transfected with the respective encoding constructs. BON1-HA and SERK4-FLAG proteins were detected using anti-HA and anti-FLAG antibodies, respectively. PIN1-HA was used as a negative control. c Split luciferase complementation (SLC) assay showing the interaction between BON1 and SERK4. PIN1 was used as a negative control. d Y2H assays showing the interaction between ZmBON1 and the ZmSERK4-KD. e SLC assay showing the interaction between ZmBON1 and ZmSERK4 in N. benthamiana leaves. ZmCDPK7 was used as a negative control. f SLC assay showing the interaction between BON1 and BRI1 in N. benthamiana leaves. PIN1 was used as a negative control. g Co-IP assay of BON1-HA and BRI1-GFP in Arabidopsis protoplasts transfected with the respective encoding constructs. BON1-HA and BRI1-GFP proteins were detected using anti-HA and anti-GFP antibodies, respectively. PIN1-HA was used as a negative control. All experiments were repeated 3 times with similar results.

Article Snippet: To examine the phosphorylation levels of native BRI1 and SERK3 in pad4-1 and bon1-1 bon2-2 bon3-3 pad4-1 after mock and 2 μM eBL treatment, total proteins were extracted from at least 50 seedling per treatment per genotype as described above and incubated with IgG Magnetic Beads (ROCKLAND, RLK00 1800) coupled with anti-BRI1 (Agrisera, AS12 1859) and anti-SERK3 (Agrisera, AS12 1858) antibodies or with anti-FLAG M2 Magnetic Beads (Sigma, M8823) for 4 h at 4 °C to respectively immunoprecipitate BRI1, SERK3, or SERK4-FLAG.

Techniques: Y2H Assay, Construct, Transformation Assay, Co-Immunoprecipitation Assay, Transfection, Negative Control, Luciferase

Journal: Nature Communications

Article Title: Copine proteins are required for brassinosteroid signaling in maize and Arabidopsis

doi: 10.1038/s41467-024-46289-6

Figure Lengend Snippet: a , c Co-IP assay of BRI1 and SERK4 ( a ) and of BRI1 and SERK3 ( c ) in the pad4-1 and bon1-1 bon2-2 bon3-3 pad4-1 ( bon123pad4-1 ) mutants without or with eBL treatment. IP was performed with FLAG beads ( a ) or α-SERK3 ( c ). Immunoprecipitated SERK4-FLAG ( a ), SERK3 ( c ) and co-immunoprecipitated BRI1 were detected with specific α-FLAG, α-SERK3, and α-BRI1 antibodies, respectively. b , d Quantification of BRI1 associated with SERK4-FLAG ( b ) or SERK3 ( d ) shown in ( a ) and ( c ), respectively. Significant differences were determined by two-tailed Student’s t -tests, asterisks indicate statistically significant differences (* P < 0.05, n = 3 biologically independent samples, ±SD). Black dots represent individual data. e Table showing the phosphopeptide sequences and the phosphorylation sites of the BR signaling components BRI1, SERK1/2/3, BKI1, BSK1, BSK5/8, BSL2, and BSL3. Phosphorylated amino acids are shown in red. f – i Phosphorylation levels of BRI1 ( f , g ), SERK3 ( h ) and SERK4 ( i ) in the pad4-1 and bon1-1 bon2-2 bon3-3 pad4-1 ( bon123pad4-1 ) mutants without or with eBL treatment. Phosphorylated proteins were detected with anti-pSer and anti-pThr antibodies. All experiments were repeated 3 times with similar results.

Article Snippet: To examine the phosphorylation levels of native BRI1 and SERK3 in pad4-1 and bon1-1 bon2-2 bon3-3 pad4-1 after mock and 2 μM eBL treatment, total proteins were extracted from at least 50 seedling per treatment per genotype as described above and incubated with IgG Magnetic Beads (ROCKLAND, RLK00 1800) coupled with anti-BRI1 (Agrisera, AS12 1859) and anti-SERK3 (Agrisera, AS12 1858) antibodies or with anti-FLAG M2 Magnetic Beads (Sigma, M8823) for 4 h at 4 °C to respectively immunoprecipitate BRI1, SERK3, or SERK4-FLAG.

Techniques: Co-Immunoprecipitation Assay, Immunoprecipitation, Two Tailed Test

Journal: Nature Communications

Article Title: Copine proteins are required for brassinosteroid signaling in maize and Arabidopsis

doi: 10.1038/s41467-024-46289-6

Figure Lengend Snippet: In the wild type, BONs interact with SERKs, which are spatially separated from BRI1. Upon BR perception, SERKs move close to BRI1 with the assistance of BONs to form the BRI1-SERKs complex. Then, BRI1 is fully activated by reciprocal phosphorylation of BRI1 and SERKs, and the BR signal is amplified to induce an efficient BR response. In the bon mutants, the interactions between BRI1 and SERKs were affected, resulting in attenuated reciprocal phosphorylation between BRI1 and SERKs, leading to an attenuated BR response.

Article Snippet: To examine the phosphorylation levels of native BRI1 and SERK3 in pad4-1 and bon1-1 bon2-2 bon3-3 pad4-1 after mock and 2 μM eBL treatment, total proteins were extracted from at least 50 seedling per treatment per genotype as described above and incubated with IgG Magnetic Beads (ROCKLAND, RLK00 1800) coupled with anti-BRI1 (Agrisera, AS12 1859) and anti-SERK3 (Agrisera, AS12 1858) antibodies or with anti-FLAG M2 Magnetic Beads (Sigma, M8823) for 4 h at 4 °C to respectively immunoprecipitate BRI1, SERK3, or SERK4-FLAG.

Techniques: Amplification

Journal: Nature Communications

Article Title: Copine proteins are required for brassinosteroid signaling in maize and Arabidopsis

doi: 10.1038/s41467-024-46289-6

Figure Lengend Snippet: a Yeast two-hybrid (Y2H) assay assessing the interaction of the A domain of BON1 with the kinase domain (KD) of SERK4, SERK1 and SERK2. The constructs were co-transformed into yeast cells and grown on selective dropout medium as indicated. b Co-IP assay of BON1-HA and SERK4-FLAG in Arabidopsis protoplasts transfected with the respective encoding constructs. BON1-HA and SERK4-FLAG proteins were detected using anti-HA and anti-FLAG antibodies, respectively. PIN1-HA was used as a negative control. c Split luciferase complementation (SLC) assay showing the interaction between BON1 and SERK4. PIN1 was used as a negative control. d Y2H assays showing the interaction between ZmBON1 and the ZmSERK4-KD. e SLC assay showing the interaction between ZmBON1 and ZmSERK4 in N. benthamiana leaves. ZmCDPK7 was used as a negative control. f SLC assay showing the interaction between BON1 and BRI1 in N. benthamiana leaves. PIN1 was used as a negative control. g Co-IP assay of BON1-HA and BRI1-GFP in Arabidopsis protoplasts transfected with the respective encoding constructs. BON1-HA and BRI1-GFP proteins were detected using anti-HA and anti-GFP antibodies, respectively. PIN1-HA was used as a negative control. All experiments were repeated 3 times with similar results.

Article Snippet: Immunoprecipitated BRI1 was detected by anti-BRI1 (Agrisera, AS12 1859, 1:5000), anti-phosphoserine (anti-pSer) (Sigma, P5747, 1:500) and anti-phosphothreonine (anti-pThr) (Cell Signaling, 9381, 1:500) antibodies, respectively.

Techniques: Y2H Assay, Construct, Transformation Assay, Co-Immunoprecipitation Assay, Transfection, Negative Control, Luciferase

Journal: Nature Communications

Article Title: Copine proteins are required for brassinosteroid signaling in maize and Arabidopsis

doi: 10.1038/s41467-024-46289-6

Figure Lengend Snippet: a , c Co-IP assay of BRI1 and SERK4 ( a ) and of BRI1 and SERK3 ( c ) in the pad4-1 and bon1-1 bon2-2 bon3-3 pad4-1 ( bon123pad4-1 ) mutants without or with eBL treatment. IP was performed with FLAG beads ( a ) or α-SERK3 ( c ). Immunoprecipitated SERK4-FLAG ( a ), SERK3 ( c ) and co-immunoprecipitated BRI1 were detected with specific α-FLAG, α-SERK3, and α-BRI1 antibodies, respectively. b , d Quantification of BRI1 associated with SERK4-FLAG ( b ) or SERK3 ( d ) shown in ( a ) and ( c ), respectively. Significant differences were determined by two-tailed Student’s t -tests, asterisks indicate statistically significant differences (* P < 0.05, n = 3 biologically independent samples, ±SD). Black dots represent individual data. e Table showing the phosphopeptide sequences and the phosphorylation sites of the BR signaling components BRI1, SERK1/2/3, BKI1, BSK1, BSK5/8, BSL2, and BSL3. Phosphorylated amino acids are shown in red. f – i Phosphorylation levels of BRI1 ( f , g ), SERK3 ( h ) and SERK4 ( i ) in the pad4-1 and bon1-1 bon2-2 bon3-3 pad4-1 ( bon123pad4-1 ) mutants without or with eBL treatment. Phosphorylated proteins were detected with anti-pSer and anti-pThr antibodies. All experiments were repeated 3 times with similar results.

Article Snippet: Immunoprecipitated BRI1 was detected by anti-BRI1 (Agrisera, AS12 1859, 1:5000), anti-phosphoserine (anti-pSer) (Sigma, P5747, 1:500) and anti-phosphothreonine (anti-pThr) (Cell Signaling, 9381, 1:500) antibodies, respectively.

Techniques: Co-Immunoprecipitation Assay, Immunoprecipitation, Two Tailed Test

Journal: Nature Communications

Article Title: Copine proteins are required for brassinosteroid signaling in maize and Arabidopsis

doi: 10.1038/s41467-024-46289-6

Figure Lengend Snippet: In the wild type, BONs interact with SERKs, which are spatially separated from BRI1. Upon BR perception, SERKs move close to BRI1 with the assistance of BONs to form the BRI1-SERKs complex. Then, BRI1 is fully activated by reciprocal phosphorylation of BRI1 and SERKs, and the BR signal is amplified to induce an efficient BR response. In the bon mutants, the interactions between BRI1 and SERKs were affected, resulting in attenuated reciprocal phosphorylation between BRI1 and SERKs, leading to an attenuated BR response.

Article Snippet: Immunoprecipitated BRI1 was detected by anti-BRI1 (Agrisera, AS12 1859, 1:5000), anti-phosphoserine (anti-pSer) (Sigma, P5747, 1:500) and anti-phosphothreonine (anti-pThr) (Cell Signaling, 9381, 1:500) antibodies, respectively.

Techniques: Amplification