Journal: Nature Communications

Article Title: NEK1-mediated retromer trafficking promotes blood–brain barrier integrity by regulating glucose metabolism and RIPK1 activation

doi: 10.1038/s41467-021-25157-7

Figure Lengend Snippet: a , b The surviving progenies (at 2 weeks and 22 weeks of age) from the following matings: 8 female Nek1 Kat2J/+ mice were mated with 4 male Nek1 Kat2J/+ mice; 8 female Nek1 Kat2J/+ ; Ripk1 D138N/D138N mice were mated with 4 male Nek1 Kat2J/+ ; Ripk1 D138N/D138N mice. The % of survival was ploted ( b ). c – f , Cerebral cortical sections from the mice (3 weeks old, female) were immunostained with anti- p -S166 RIPK1 ( c ) and anti-p-S345 MLKL ( e ). The fluorescent signals of p-S166 RIPK1 ( d ) and p-S345 MLKL ( f ) were quantified. Mean ± SD. n = 3 sections per group for p-S166 RIPK1. n = 6 sections per group for p-S345 MLKL. Bar = 50 µm. One-way ANOVA with Dunnett’s test. g Mice (3 weeks old) were inraperitoneally injected with 2% (w/v) Evans blue solution (70 mg/kg) and followed by PBS perfusion. Extravasated Evans blue levels in total brain lysates were measured by a plate reader. Mean ± SD. n = 3 mice per group. One-way ANOVA with Dunnett’s test. h , i Mice (3 weeks old) were intravenously injected with 10kD-tetramethyrhodamine (TMR)-labeled Dextran and then sacrificed. The cryo-sections of cerebral cortex were imaged by confocal microscope. Bar = 20 µm. Fluorescent intensity of diffused dextran around cerebral vessels was quantified using ImageJ. The fluorescent intensity of all groups was nomalized to Nek1 +/+ mice (mean ± SD, n = 4 sections per group) and graphed in ( i ). One-way ANOVA with Dunnett’s test. j , k Representative images of Gr1 immunohistostaining (brown) of cerebral cortex and thalamus sections (3 weeks old mice). Bar = 20 µm.Number of Gr1 positive cells per sections were presented as mean± SD. n = 3 sections per group. One-way ANOVA with Dunnett’s test.

Article Snippet: Antibodies against the following proteins were used for western blot analysis: NEK1 (Abcam #ab229489, 1:1000), NEK1 (Santa Cruz #sc-398813, 1:500), p-S166 RIPK1 (CST #31122, 1:1000), p-S166 RIPK1 (Arigo # ARG66476, 1:1000), RIPK1 (CST #3493, 1:2000), A20 (CST, #5630, 1:1000), TNFR1 (CST #13377, 1:1000), IκBα (CST #9242, 1:2000), p-S32/36 IκBα (CST, #9246, 1:2000), FLAG (Sigma-Aldrich #F3165, 1:2000), HA (CST #3724, 1:2000), α-synuclein (Invitrogen #AHB0261, 1:2000), p-S345 MLKL (CST #91689, 1:1000), MLKL (CST #37705, 1:1000), p-T231/S232 RIPK3 (CST #57220, 1:1000), RIPK3 (CST #13526, 1:1000), p-S79 acetyl-CoA carboxylase (CST #3661, 1:1000), acetyl-CoA carboxylase (CST #3662, 1:1000), SQSTM1/p62 (CST #5114, 1:5000), LAMP1 (Santa Cruz #sc-19992, 1:5000), p-Ser(EMD Millipore #05-1000, 1:2000).

Techniques: Injection, Labeling, Microscopy

Journal: Nature Communications

Article Title: NEK1-mediated retromer trafficking promotes blood–brain barrier integrity by regulating glucose metabolism and RIPK1 activation

doi: 10.1038/s41467-021-25157-7

Figure Lengend Snippet: a Nek1 +/+ and Nek1 Kat2J/Kat2J MEFs were pre-treated with 20 mM acetyl-l-carnitine for 18 h and then treated with1 ng/mL TNF + 500 nM 5z7 to induce RDA for 3, 4, and 6 h. Cell death was measured by ToxiLight. Mean ± SD, n=3 biological independent samples. One-way ANOVA with Dunnett’s test. b Nek1 +/+ and Nek1 Kat2J/Kat2J MEFs were pre-treated with indicated concentrations of sodium octonate for 18 h and then treated with 1 ng/mL TNF + 500 nM 5z7 to induce RDA for 6 h. The % of cell death was measured by ToxiLight. Mean ± SD. n = 3 biological independent repeats. One-way ANOVA with Dunnett’s test. c – e The sizes of lysosomes in Nek1 +/+ and Nek1 Kat2J/Kat2J MEFs were determined with a biomarker LAMP1 by immunostaining ( c ) and quantified with ImageJ for sizes ( d ) and density ( e ). n = 6 cells per group. Bar = 10 µm.Data are presented as mean ± SEM. Unpaired two-tailed Student’s t-test. f , Whole-cell lysates of Nek1 +/+ and Nek1 Kat2J/Kat2J MEFs were analyzed by western blotting with indicated antibodies. Uncropped blots in the Source Data file. g Nek1 +/+ and Nek1 Kat2J/Kat2J MEFs were stained with 50 nM MitoTracker-Green for 30 min and imaged by fluorescent microscopy. The length of mitochondria was quantified with ImageJ. Mean ± SD, n = 12 cells for each group. Bar = 10 µm. Unpaired two-tailed Student’s t-test. h , Nek1 +/+ and Nek1 Kat2J/Kat2J MEFs were treated with indicated concentration of acetyl-l-carnitine for 24 h, and then treated with 50 nM MitoTracker-Green for 30 min. The images were acquired by confocal microscopy. The average branch lengthes of mitochondria were quantified with ImageJ ( n = 5 cells per group). The data are presented as mean ± SD. One-way ANOVA with Dunnett’s test. Bar = 10 µm. i WT and Nek1 Kat2J/Kat2J MEFs were treated with 0, 1 mM, 5 mM, 10 mM, and 20 mM acetyl-l-carnitine for 20 hours, then lysed with SDS buffer. Uncropped blots in the Source Data file. j Nek1 +/+ and Nek1 Kat2J/Kat2J MEFs were pre-treated with 20 mM acetyl-l-carnitine for 18 h and then treated with 10 ng/mL TNF + 50 nM SM164 + 20 µM zVAD.fmk for indicated periods of time. The cell lysates lysed with NP40 buffer and with 6 M urea were analyzed by western blotting using indicated abs. Uncropped blots in the Source Data file.

Article Snippet: Antibodies against the following proteins were used for western blot analysis: NEK1 (Abcam #ab229489, 1:1000), NEK1 (Santa Cruz #sc-398813, 1:500), p-S166 RIPK1 (CST #31122, 1:1000), p-S166 RIPK1 (Arigo # ARG66476, 1:1000), RIPK1 (CST #3493, 1:2000), A20 (CST, #5630, 1:1000), TNFR1 (CST #13377, 1:1000), IκBα (CST #9242, 1:2000), p-S32/36 IκBα (CST, #9246, 1:2000), FLAG (Sigma-Aldrich #F3165, 1:2000), HA (CST #3724, 1:2000), α-synuclein (Invitrogen #AHB0261, 1:2000), p-S345 MLKL (CST #91689, 1:1000), MLKL (CST #37705, 1:1000), p-T231/S232 RIPK3 (CST #57220, 1:1000), RIPK3 (CST #13526, 1:1000), p-S79 acetyl-CoA carboxylase (CST #3661, 1:1000), acetyl-CoA carboxylase (CST #3662, 1:1000), SQSTM1/p62 (CST #5114, 1:5000), LAMP1 (Santa Cruz #sc-19992, 1:5000), p-Ser(EMD Millipore #05-1000, 1:2000).

Techniques: Biomarker Discovery, Immunostaining, Two Tailed Test, Western Blot, Staining, Microscopy, Concentration Assay, Confocal Microscopy

Journal: Nature Communications

Article Title: NEK1-mediated retromer trafficking promotes blood–brain barrier integrity by regulating glucose metabolism and RIPK1 activation

doi: 10.1038/s41467-021-25157-7

Figure Lengend Snippet: a , b The surviving progenies (at 2 weeks and 22 weeks of age) from the following matings: 8 female Nek1 Kat2J/+ mice were mated with 4 male Nek1 Kat2J/+ mice; 8 female Nek1 Kat2J/+ ; Ripk1 D138N/D138N mice were mated with 4 male Nek1 Kat2J/+ ; Ripk1 D138N/D138N mice. The % of survival was ploted ( b ). c – f , Cerebral cortical sections from the mice (3 weeks old, female) were immunostained with anti- p -S166 RIPK1 ( c ) and anti-p-S345 MLKL ( e ). The fluorescent signals of p-S166 RIPK1 ( d ) and p-S345 MLKL ( f ) were quantified. Mean ± SD. n = 3 sections per group for p-S166 RIPK1. n = 6 sections per group for p-S345 MLKL. Bar = 50 µm. One-way ANOVA with Dunnett’s test. g Mice (3 weeks old) were inraperitoneally injected with 2% (w/v) Evans blue solution (70 mg/kg) and followed by PBS perfusion. Extravasated Evans blue levels in total brain lysates were measured by a plate reader. Mean ± SD. n = 3 mice per group. One-way ANOVA with Dunnett’s test. h , i Mice (3 weeks old) were intravenously injected with 10kD-tetramethyrhodamine (TMR)-labeled Dextran and then sacrificed. The cryo-sections of cerebral cortex were imaged by confocal microscope. Bar = 20 µm. Fluorescent intensity of diffused dextran around cerebral vessels was quantified using ImageJ. The fluorescent intensity of all groups was nomalized to Nek1 +/+ mice (mean ± SD, n = 4 sections per group) and graphed in ( i ). One-way ANOVA with Dunnett’s test. j , k Representative images of Gr1 immunohistostaining (brown) of cerebral cortex and thalamus sections (3 weeks old mice). Bar = 20 µm.Number of Gr1 positive cells per sections were presented as mean± SD. n = 3 sections per group. One-way ANOVA with Dunnett’s test.

Article Snippet: Antibodies against the following proteins were used for western blot analysis: NEK1 (Abcam #ab229489, 1:1000), NEK1 (Santa Cruz #sc-398813, 1:500), p-S166 RIPK1 (CST #31122, 1:1000), p-S166 RIPK1 (Arigo # ARG66476, 1:1000), RIPK1 (CST #3493, 1:2000), A20 (CST, #5630, 1:1000), TNFR1 (CST #13377, 1:1000), IκBα (CST #9242, 1:2000), p-S32/36 IκBα (CST, #9246, 1:2000), FLAG (Sigma-Aldrich #F3165, 1:2000), HA (CST #3724, 1:2000), α-synuclein (Invitrogen #AHB0261, 1:2000), p-S345 MLKL (CST #91689, 1:1000), MLKL (CST #37705, 1:1000), p-T231/S232 RIPK3 (CST #57220, 1:1000), RIPK3 (CST #13526, 1:1000), p-S79 acetyl-CoA carboxylase (CST #3661, 1:1000), acetyl-CoA carboxylase (CST #3662, 1:1000), SQSTM1/p62 (CST #5114, 1:5000), LAMP1 (Santa Cruz #sc-19992, 1:5000), p-Ser(EMD Millipore #05-1000, 1:2000).

Techniques: Injection, Labeling, Microscopy

Journal: Nature Communications

Article Title: NEK1-mediated retromer trafficking promotes blood–brain barrier integrity by regulating glucose metabolism and RIPK1 activation

doi: 10.1038/s41467-021-25157-7

Figure Lengend Snippet: a , b The surviving progenies (at 2 weeks and 22 weeks of age) from the following matings: 8 female Nek1 Kat2J/+ mice were mated with 4 male Nek1 Kat2J/+ mice; 8 female Nek1 Kat2J/+ ; Ripk1 D138N/D138N mice were mated with 4 male Nek1 Kat2J/+ ; Ripk1 D138N/D138N mice. The % of survival was ploted ( b ). c – f , Cerebral cortical sections from the mice (3 weeks old, female) were immunostained with anti- p -S166 RIPK1 ( c ) and anti-p-S345 MLKL ( e ). The fluorescent signals of p-S166 RIPK1 ( d ) and p-S345 MLKL ( f ) were quantified. Mean ± SD. n = 3 sections per group for p-S166 RIPK1. n = 6 sections per group for p-S345 MLKL. Bar = 50 µm. One-way ANOVA with Dunnett’s test. g Mice (3 weeks old) were inraperitoneally injected with 2% (w/v) Evans blue solution (70 mg/kg) and followed by PBS perfusion. Extravasated Evans blue levels in total brain lysates were measured by a plate reader. Mean ± SD. n = 3 mice per group. One-way ANOVA with Dunnett’s test. h , i Mice (3 weeks old) were intravenously injected with 10kD-tetramethyrhodamine (TMR)-labeled Dextran and then sacrificed. The cryo-sections of cerebral cortex were imaged by confocal microscope. Bar = 20 µm. Fluorescent intensity of diffused dextran around cerebral vessels was quantified using ImageJ. The fluorescent intensity of all groups was nomalized to Nek1 +/+ mice (mean ± SD, n = 4 sections per group) and graphed in ( i ). One-way ANOVA with Dunnett’s test. j , k Representative images of Gr1 immunohistostaining (brown) of cerebral cortex and thalamus sections (3 weeks old mice). Bar = 20 µm.Number of Gr1 positive cells per sections were presented as mean± SD. n = 3 sections per group. One-way ANOVA with Dunnett’s test.

Article Snippet: Antibodies against the following proteins were used for western blot analysis: NEK1 (Abcam #ab229489, 1:1000), NEK1 (Santa Cruz #sc-398813, 1:500), p-S166 RIPK1 (CST #31122, 1:1000), p-S166 RIPK1 (Arigo # ARG66476, 1:1000), RIPK1 (CST #3493, 1:2000), A20 (CST, #5630, 1:1000), TNFR1 (CST #13377, 1:1000), IκBα (CST #9242, 1:2000), p-S32/36 IκBα (CST, #9246, 1:2000), FLAG (Sigma-Aldrich #F3165, 1:2000), HA (CST #3724, 1:2000), α-synuclein (Invitrogen #AHB0261, 1:2000), p-S345 MLKL (CST #91689, 1:1000), MLKL (CST #37705, 1:1000), p-T231/S232 RIPK3 (CST #57220, 1:1000), RIPK3 (CST #13526, 1:1000), p-S79 acetyl-CoA carboxylase (CST #3661, 1:1000), acetyl-CoA carboxylase (CST #3662, 1:1000), SQSTM1/p62 (CST #5114, 1:5000), LAMP1 (Santa Cruz #sc-19992, 1:5000), p-Ser(EMD Millipore #05-1000, 1:2000).

Techniques: Injection, Labeling, Microscopy

Journal: Nature Communications

Article Title: NEK1-mediated retromer trafficking promotes blood–brain barrier integrity by regulating glucose metabolism and RIPK1 activation

doi: 10.1038/s41467-021-25157-7

Figure Lengend Snippet: a WT and Nek1 Kat2J/Kat2J MEFs were treated with FLAG-TNF 50 ng/mL for indicated periods of time. Whole cell lysates and complex I of the treated MEFs were analyzed by western blotting with indicated antibodies. Uncropped blots in the Source Data file. b WT and Nek1 Kat2J/Kat2J MEFs were treated with FLAG-TNF 50 ng/mL for indicated periods of time and then lysed with NP40 buffer. The cell lysates were immunoprecipitated with anti-FLAG antibody conjugated agarose (1 st IP). The immunoprecipitated proteins were eluted and immunoprecipitated with anti-K63 ubiquitin antibody (2 nd IP). Uncropped blots in the Source Data file. c , d WT and Nek1 Kat2J/Kat2J MEFs were transfected with expression vectors of HA-NEK1, HA-NEK1 D146N or vector for 72 h and then the cells were lysed and analyzed by western blotting using indicated abs ( c ) and quantified with ImageJ ( d ). Uncropped blots in the Source Data file. n=3 replicates for each group. One-way ANOVA, Dunnett’s test. e Nek1 +/+ and Nek1 Kat2J/Kat2J MEFs were treated with 25 μM chloroquine (CQ) + 5 μg/mL E64d for 12 and 24 h. Uncropped blots in the Source Data file. f Nek1 +/+ and Nek1 Kat2J/Kat2J MEFs were treated with 10 μM MG132 for 3 and 6 h. The levels of A20 were analyzed by western blotting with indicated antibodies and quantified using ImageJ (bottom). Uncropped blots in the Source Data file. g Nek1 +/+ and Nek1 Kat2J/Kat2J MEFs were pretreated with 10 μM Nec-1s for 30 min and then treated with 1 ng/mL TNF + 500 nM 5z7 for indicated time. Cell lysates were analyzed by western blotting with indicated antibodies. Uncropped blots in the Source Data file. h Nek1 +/+ and Nek1 Kat2J/Kat2J MEFs were pre-treated with 10 μM Nec-1s + 50 nM SM164 for 30 min and then treated with 10 ng/mL TNF + 20 µM zVAD.fmk for indicated periods of time. Cell lysates were analyzed by western blotting with indicated antibodies. Uncropped blots in the Source Data file. i Cerebromicrovessles were isolated from the brains of Nek1 +/+ ; Ripk1 D138N/D138N and Nek1 Kat2J/Kat2J ; Ripk1 D138N/D138N mice. The brains of two 10-month-old mice of each genotype were pooled together for the isolation of cerebromicrovessles which were analyzed by western blotting and quantified using ImageJ. n = 3 repeated blots. Data are presented as mean ± SD. Two-tailed Student’s t test. Uncropped blots in the Source Data file.

Article Snippet: Antibodies against the following proteins were used for western blot analysis: NEK1 (Abcam #ab229489, 1:1000), NEK1 (Santa Cruz #sc-398813, 1:500), p-S166 RIPK1 (CST #31122, 1:1000), p-S166 RIPK1 (Arigo # ARG66476, 1:1000), RIPK1 (CST #3493, 1:2000), A20 (CST, #5630, 1:1000), TNFR1 (CST #13377, 1:1000), IκBα (CST #9242, 1:2000), p-S32/36 IκBα (CST, #9246, 1:2000), FLAG (Sigma-Aldrich #F3165, 1:2000), HA (CST #3724, 1:2000), α-synuclein (Invitrogen #AHB0261, 1:2000), p-S345 MLKL (CST #91689, 1:1000), MLKL (CST #37705, 1:1000), p-T231/S232 RIPK3 (CST #57220, 1:1000), RIPK3 (CST #13526, 1:1000), p-S79 acetyl-CoA carboxylase (CST #3661, 1:1000), acetyl-CoA carboxylase (CST #3662, 1:1000), SQSTM1/p62 (CST #5114, 1:5000), LAMP1 (Santa Cruz #sc-19992, 1:5000), p-Ser(EMD Millipore #05-1000, 1:2000).

Techniques: Western Blot, Immunoprecipitation, Ubiquitin Proteomics, Transfection, Expressing, Plasmid Preparation, Isolation, Two Tailed Test

Journal: Nature Communications

Article Title: NEK1-mediated retromer trafficking promotes blood–brain barrier integrity by regulating glucose metabolism and RIPK1 activation

doi: 10.1038/s41467-021-25157-7

Figure Lengend Snippet: a , b Cerebral cortical sections from mice (3 weeks old) with indicated genotypes were stained with Thioflavin S for protein aggregates and quantified ( b ). Mean ± SD. n = 4 sections. One-way ANOVA with Dunnett’s test. Bar = 50 µm. c , d Cerebral cortical sections of mice (3 weeks old) with indicated genotypes were immunostained with anti-α-synuclein(red) and anti-NeuN(green); and the fluorescent intensity of α-synuclein in each NeuN positive cells was quantified ( d ). Bar = 50 µm. n = 3 sections per group. Data are presented as mean ± SEM. One-way ANOVA with Dunnett’s test. e Total cortical lysates from Nek1 +/+ mice, Nek1 Kat2J/Kat2J mice, Nek1 +/+ ; Ripk1 D138N/D138N mice and Nek1 Kat2J/Kat2J ; Ripk1 D138N/D138N mice were analyzed by western blotting with indicated antibodies. Uncropped blots in the Source Data file. f , g The mRNAs from spinal cords ( f ) or brains ( g ) mice with indicated genotypes ( n = 3 per group, 3 weeks old) were extracted and quantified for the levels of indicated cytokines by qPCR. Data are presented as mean ± SD. One-way ANOVA with Dunnett’s test.

Article Snippet: Antibodies against the following proteins were used for western blot analysis: NEK1 (Abcam #ab229489, 1:1000), NEK1 (Santa Cruz #sc-398813, 1:500), p-S166 RIPK1 (CST #31122, 1:1000), p-S166 RIPK1 (Arigo # ARG66476, 1:1000), RIPK1 (CST #3493, 1:2000), A20 (CST, #5630, 1:1000), TNFR1 (CST #13377, 1:1000), IκBα (CST #9242, 1:2000), p-S32/36 IκBα (CST, #9246, 1:2000), FLAG (Sigma-Aldrich #F3165, 1:2000), HA (CST #3724, 1:2000), α-synuclein (Invitrogen #AHB0261, 1:2000), p-S345 MLKL (CST #91689, 1:1000), MLKL (CST #37705, 1:1000), p-T231/S232 RIPK3 (CST #57220, 1:1000), RIPK3 (CST #13526, 1:1000), p-S79 acetyl-CoA carboxylase (CST #3661, 1:1000), acetyl-CoA carboxylase (CST #3662, 1:1000), SQSTM1/p62 (CST #5114, 1:5000), LAMP1 (Santa Cruz #sc-19992, 1:5000), p-Ser(EMD Millipore #05-1000, 1:2000).

Techniques: Staining, Western Blot

Journal: Nature Communications

Article Title: NEK1-mediated retromer trafficking promotes blood–brain barrier integrity by regulating glucose metabolism and RIPK1 activation

doi: 10.1038/s41467-021-25157-7

Figure Lengend Snippet: a Whole brain lysates of Nek1 +/+ mice, Nek1 Kat2J/Kat2J mice, Nek1 +/+ ;Ripk1 D138N/D138N mice and Nek1 Kat2J/Kat2J ; Ripk1 D138N/D138N mice were analyzed by western blotting with indicated antibodies. Uncropped blots in the Source Data file. b The integrity of BBB in Nek1 +/+ newborn mice and Nek1 Kat2J/Kat2J newborn mice, fed with control diet or ketogenic diet since the pregnancy of their mothers, was assessed by intraperitoneal injection of Evans blue and the cerebral retention of Evans blue was assessed at 24 h after the injection with PBS perfusion. n = 3 mice for each group. One-way ANOVA with Dunnett’s test. c The postnatal survival of Nek1 Kat2J/Kat2J mice on control diet or ketogenic diet. N = 7 for each group. Unpaired two-tailed Student’s t-test. d , e Gr1 immunohistostaining (brown) of cerebral cortex and thalamus sections from the brains of Nek1 Kat2J/Kat2J or WT mice (3 weeks old) fed with ketogenic diet or control diet ( d ) and the numbers of Gr1 positive cells per sections were quantified ( e ). Bar = 20 µm. n = 3 sections per group. One-way ANOVA with Dunnett’s test. f – g Cerebromicrovessles were isolated from the brains of 3 Nek1 +/+ mice and 3 Nek1 Kat2J/Kat2J mice fed with control diet or ketogenic diet. The lysates of isolated cerebromicrovessles of each genotypes were analyzed by western blotting with indicated antibodies ( f ) and quantified using ImageJ ( g ). n = 3 replicates for each group. One-way ANOVA with Dunnett’s test. Uncropped blots in the Source Data file. h - i The mRNAs isolated from brains ( h ) and spinal cords ( i ) of Nek1 +/+ mice and Nek1 Kat2J/Kat2J mice fed with control diet or ketogenic diet (n=3 per group, 3 weeks old) were quantified for the levels of indicated cytokines by qPCR. Data are presented as mean ± SD. One-way ANOVA with Dunnett’s test.

Article Snippet: Antibodies against the following proteins were used for western blot analysis: NEK1 (Abcam #ab229489, 1:1000), NEK1 (Santa Cruz #sc-398813, 1:500), p-S166 RIPK1 (CST #31122, 1:1000), p-S166 RIPK1 (Arigo # ARG66476, 1:1000), RIPK1 (CST #3493, 1:2000), A20 (CST, #5630, 1:1000), TNFR1 (CST #13377, 1:1000), IκBα (CST #9242, 1:2000), p-S32/36 IκBα (CST, #9246, 1:2000), FLAG (Sigma-Aldrich #F3165, 1:2000), HA (CST #3724, 1:2000), α-synuclein (Invitrogen #AHB0261, 1:2000), p-S345 MLKL (CST #91689, 1:1000), MLKL (CST #37705, 1:1000), p-T231/S232 RIPK3 (CST #57220, 1:1000), RIPK3 (CST #13526, 1:1000), p-S79 acetyl-CoA carboxylase (CST #3661, 1:1000), acetyl-CoA carboxylase (CST #3662, 1:1000), SQSTM1/p62 (CST #5114, 1:5000), LAMP1 (Santa Cruz #sc-19992, 1:5000), p-Ser(EMD Millipore #05-1000, 1:2000).

Techniques: Western Blot, Control, Injection, Two Tailed Test, Isolation

Journal: Nature Communications

Article Title: NEK1-mediated retromer trafficking promotes blood–brain barrier integrity by regulating glucose metabolism and RIPK1 activation

doi: 10.1038/s41467-021-25157-7

Figure Lengend Snippet: Phosphorylation of S302/S304 VPS26B by NEK1 modulates the recycling of SNX27-retromer and SNX2-retromer that deliver proteins from endosomes to cell surface membrane and lysosomes. NEK1 deficiency blocks the recycling of SNX27-retromer from cell surface to endosomes. Since SNX27-retromer is important for delivering GLUT1 to the cell surface membrane, NEK1 deficiency leads to reduced GLUT1 levels at the cell surface membrane which reduces glucose uptake. Reduced glucose uptake disrupts mitochondrial metabolism and reduces cellular levels of acetyl-CoA. Reduced acetyl-CoA inhibits the acetylation of A20 which promotes its lysosomal degradation. Reduced levels of A20 sensitizes to the activation of RIPK1-dependent cell death and inflammation which can be inhibited by Nec-1s and also by ketogenic diet which provides alternate source of acetyl-CoA to restore cellular levels of A20.

Article Snippet: Antibodies against the following proteins were used for western blot analysis: NEK1 (Abcam #ab229489, 1:1000), NEK1 (Santa Cruz #sc-398813, 1:500), p-S166 RIPK1 (CST #31122, 1:1000), p-S166 RIPK1 (Arigo # ARG66476, 1:1000), RIPK1 (CST #3493, 1:2000), A20 (CST, #5630, 1:1000), TNFR1 (CST #13377, 1:1000), IκBα (CST #9242, 1:2000), p-S32/36 IκBα (CST, #9246, 1:2000), FLAG (Sigma-Aldrich #F3165, 1:2000), HA (CST #3724, 1:2000), α-synuclein (Invitrogen #AHB0261, 1:2000), p-S345 MLKL (CST #91689, 1:1000), MLKL (CST #37705, 1:1000), p-T231/S232 RIPK3 (CST #57220, 1:1000), RIPK3 (CST #13526, 1:1000), p-S79 acetyl-CoA carboxylase (CST #3661, 1:1000), acetyl-CoA carboxylase (CST #3662, 1:1000), SQSTM1/p62 (CST #5114, 1:5000), LAMP1 (Santa Cruz #sc-19992, 1:5000), p-Ser(EMD Millipore #05-1000, 1:2000).

Techniques: Phospho-proteomics, Membrane, Activation Assay

Journal: Nature Communications

Article Title: NEK1-mediated retromer trafficking promotes blood–brain barrier integrity by regulating glucose metabolism and RIPK1 activation

doi: 10.1038/s41467-021-25157-7

Figure Lengend Snippet: a , b The surviving progenies (at 2 weeks and 22 weeks of age) from the following matings: 8 female Nek1 Kat2J/+ mice were mated with 4 male Nek1 Kat2J/+ mice; 8 female Nek1 Kat2J/+ ; Ripk1 D138N/D138N mice were mated with 4 male Nek1 Kat2J/+ ; Ripk1 D138N/D138N mice. The % of survival was ploted ( b ). c – f , Cerebral cortical sections from the mice (3 weeks old, female) were immunostained with anti- p -S166 RIPK1 ( c ) and anti-p-S345 MLKL ( e ). The fluorescent signals of p-S166 RIPK1 ( d ) and p-S345 MLKL ( f ) were quantified. Mean ± SD. n = 3 sections per group for p-S166 RIPK1. n = 6 sections per group for p-S345 MLKL. Bar = 50 µm. One-way ANOVA with Dunnett’s test. g Mice (3 weeks old) were inraperitoneally injected with 2% (w/v) Evans blue solution (70 mg/kg) and followed by PBS perfusion. Extravasated Evans blue levels in total brain lysates were measured by a plate reader. Mean ± SD. n = 3 mice per group. One-way ANOVA with Dunnett’s test. h , i Mice (3 weeks old) were intravenously injected with 10kD-tetramethyrhodamine (TMR)-labeled Dextran and then sacrificed. The cryo-sections of cerebral cortex were imaged by confocal microscope. Bar = 20 µm. Fluorescent intensity of diffused dextran around cerebral vessels was quantified using ImageJ. The fluorescent intensity of all groups was nomalized to Nek1 +/+ mice (mean ± SD, n = 4 sections per group) and graphed in ( i ). One-way ANOVA with Dunnett’s test. j , k Representative images of Gr1 immunohistostaining (brown) of cerebral cortex and thalamus sections (3 weeks old mice). Bar = 20 µm.Number of Gr1 positive cells per sections were presented as mean± SD. n = 3 sections per group. One-way ANOVA with Dunnett’s test.

Article Snippet: Antibodies against the following proteins were used for western blot analysis: NEK1 (Abcam #ab229489, 1:1000), NEK1 (Santa Cruz #sc-398813, 1:500), p-S166 RIPK1 (CST #31122, 1:1000), p-S166 RIPK1 (Arigo # ARG66476, 1:1000), RIPK1 (CST #3493, 1:2000), A20 (CST, #5630, 1:1000), TNFR1 (CST #13377, 1:1000), IκBα (CST #9242, 1:2000), p-S32/36 IκBα (CST, #9246, 1:2000), FLAG (Sigma-Aldrich #F3165, 1:2000), HA (CST #3724, 1:2000), α-synuclein (Invitrogen #AHB0261, 1:2000), p-S345 MLKL (CST #91689, 1:1000), MLKL (CST #37705, 1:1000), p-T231/S232 RIPK3 (CST #57220, 1:1000), RIPK3 (CST #13526, 1:1000), p-S79 acetyl-CoA carboxylase (CST #3661, 1:1000), acetyl-CoA carboxylase (CST #3662, 1:1000), SQSTM1/p62 (CST #5114, 1:5000), LAMP1 (Santa Cruz #sc-19992, 1:5000), p-Ser(EMD Millipore #05-1000, 1:2000).

Techniques: Injection, Labeling, Microscopy

Journal: Nature Communications

Article Title: NEK1-mediated retromer trafficking promotes blood–brain barrier integrity by regulating glucose metabolism and RIPK1 activation

doi: 10.1038/s41467-021-25157-7

Figure Lengend Snippet: a WT and Nek1 Kat2J/Kat2J MEFs were treated with FLAG-TNF 50 ng/mL for indicated periods of time. Whole cell lysates and complex I of the treated MEFs were analyzed by western blotting with indicated antibodies. Uncropped blots in the Source Data file. b WT and Nek1 Kat2J/Kat2J MEFs were treated with FLAG-TNF 50 ng/mL for indicated periods of time and then lysed with NP40 buffer. The cell lysates were immunoprecipitated with anti-FLAG antibody conjugated agarose (1 st IP). The immunoprecipitated proteins were eluted and immunoprecipitated with anti-K63 ubiquitin antibody (2 nd IP). Uncropped blots in the Source Data file. c , d WT and Nek1 Kat2J/Kat2J MEFs were transfected with expression vectors of HA-NEK1, HA-NEK1 D146N or vector for 72 h and then the cells were lysed and analyzed by western blotting using indicated abs ( c ) and quantified with ImageJ ( d ). Uncropped blots in the Source Data file. n=3 replicates for each group. One-way ANOVA, Dunnett’s test. e Nek1 +/+ and Nek1 Kat2J/Kat2J MEFs were treated with 25 μM chloroquine (CQ) + 5 μg/mL E64d for 12 and 24 h. Uncropped blots in the Source Data file. f Nek1 +/+ and Nek1 Kat2J/Kat2J MEFs were treated with 10 μM MG132 for 3 and 6 h. The levels of A20 were analyzed by western blotting with indicated antibodies and quantified using ImageJ (bottom). Uncropped blots in the Source Data file. g Nek1 +/+ and Nek1 Kat2J/Kat2J MEFs were pretreated with 10 μM Nec-1s for 30 min and then treated with 1 ng/mL TNF + 500 nM 5z7 for indicated time. Cell lysates were analyzed by western blotting with indicated antibodies. Uncropped blots in the Source Data file. h Nek1 +/+ and Nek1 Kat2J/Kat2J MEFs were pre-treated with 10 μM Nec-1s + 50 nM SM164 for 30 min and then treated with 10 ng/mL TNF + 20 µM zVAD.fmk for indicated periods of time. Cell lysates were analyzed by western blotting with indicated antibodies. Uncropped blots in the Source Data file. i Cerebromicrovessles were isolated from the brains of Nek1 +/+ ; Ripk1 D138N/D138N and Nek1 Kat2J/Kat2J ; Ripk1 D138N/D138N mice. The brains of two 10-month-old mice of each genotype were pooled together for the isolation of cerebromicrovessles which were analyzed by western blotting and quantified using ImageJ. n = 3 repeated blots. Data are presented as mean ± SD. Two-tailed Student’s t test. Uncropped blots in the Source Data file.

Article Snippet: Antibodies against the following proteins were used for western blot analysis: NEK1 (Abcam #ab229489, 1:1000), NEK1 (Santa Cruz #sc-398813, 1:500), p-S166 RIPK1 (CST #31122, 1:1000), p-S166 RIPK1 (Arigo # ARG66476, 1:1000), RIPK1 (CST #3493, 1:2000), A20 (CST, #5630, 1:1000), TNFR1 (CST #13377, 1:1000), IκBα (CST #9242, 1:2000), p-S32/36 IκBα (CST, #9246, 1:2000), FLAG (Sigma-Aldrich #F3165, 1:2000), HA (CST #3724, 1:2000), α-synuclein (Invitrogen #AHB0261, 1:2000), p-S345 MLKL (CST #91689, 1:1000), MLKL (CST #37705, 1:1000), p-T231/S232 RIPK3 (CST #57220, 1:1000), RIPK3 (CST #13526, 1:1000), p-S79 acetyl-CoA carboxylase (CST #3661, 1:1000), acetyl-CoA carboxylase (CST #3662, 1:1000), SQSTM1/p62 (CST #5114, 1:5000), LAMP1 (Santa Cruz #sc-19992, 1:5000), p-Ser(EMD Millipore #05-1000, 1:2000).

Techniques: Western Blot, Immunoprecipitation, Ubiquitin Proteomics, Transfection, Expressing, Plasmid Preparation, Isolation, Two Tailed Test

Journal: Nature Communications

Article Title: NEK1-mediated retromer trafficking promotes blood–brain barrier integrity by regulating glucose metabolism and RIPK1 activation

doi: 10.1038/s41467-021-25157-7

Figure Lengend Snippet: a , b Cerebral cortical sections from mice (3 weeks old) with indicated genotypes were stained with Thioflavin S for protein aggregates and quantified ( b ). Mean ± SD. n = 4 sections. One-way ANOVA with Dunnett’s test. Bar = 50 µm. c , d Cerebral cortical sections of mice (3 weeks old) with indicated genotypes were immunostained with anti-α-synuclein(red) and anti-NeuN(green); and the fluorescent intensity of α-synuclein in each NeuN positive cells was quantified ( d ). Bar = 50 µm. n = 3 sections per group. Data are presented as mean ± SEM. One-way ANOVA with Dunnett’s test. e Total cortical lysates from Nek1 +/+ mice, Nek1 Kat2J/Kat2J mice, Nek1 +/+ ; Ripk1 D138N/D138N mice and Nek1 Kat2J/Kat2J ; Ripk1 D138N/D138N mice were analyzed by western blotting with indicated antibodies. Uncropped blots in the Source Data file. f , g The mRNAs from spinal cords ( f ) or brains ( g ) mice with indicated genotypes ( n = 3 per group, 3 weeks old) were extracted and quantified for the levels of indicated cytokines by qPCR. Data are presented as mean ± SD. One-way ANOVA with Dunnett’s test.

Article Snippet: Antibodies against the following proteins were used for western blot analysis: NEK1 (Abcam #ab229489, 1:1000), NEK1 (Santa Cruz #sc-398813, 1:500), p-S166 RIPK1 (CST #31122, 1:1000), p-S166 RIPK1 (Arigo # ARG66476, 1:1000), RIPK1 (CST #3493, 1:2000), A20 (CST, #5630, 1:1000), TNFR1 (CST #13377, 1:1000), IκBα (CST #9242, 1:2000), p-S32/36 IκBα (CST, #9246, 1:2000), FLAG (Sigma-Aldrich #F3165, 1:2000), HA (CST #3724, 1:2000), α-synuclein (Invitrogen #AHB0261, 1:2000), p-S345 MLKL (CST #91689, 1:1000), MLKL (CST #37705, 1:1000), p-T231/S232 RIPK3 (CST #57220, 1:1000), RIPK3 (CST #13526, 1:1000), p-S79 acetyl-CoA carboxylase (CST #3661, 1:1000), acetyl-CoA carboxylase (CST #3662, 1:1000), SQSTM1/p62 (CST #5114, 1:5000), LAMP1 (Santa Cruz #sc-19992, 1:5000), p-Ser(EMD Millipore #05-1000, 1:2000).

Techniques: Staining, Western Blot

Journal: Nature Communications

Article Title: NEK1-mediated retromer trafficking promotes blood–brain barrier integrity by regulating glucose metabolism and RIPK1 activation

doi: 10.1038/s41467-021-25157-7

Figure Lengend Snippet: a Whole brain lysates of Nek1 +/+ mice, Nek1 Kat2J/Kat2J mice, Nek1 +/+ ;Ripk1 D138N/D138N mice and Nek1 Kat2J/Kat2J ; Ripk1 D138N/D138N mice were analyzed by western blotting with indicated antibodies. Uncropped blots in the Source Data file. b The integrity of BBB in Nek1 +/+ newborn mice and Nek1 Kat2J/Kat2J newborn mice, fed with control diet or ketogenic diet since the pregnancy of their mothers, was assessed by intraperitoneal injection of Evans blue and the cerebral retention of Evans blue was assessed at 24 h after the injection with PBS perfusion. n = 3 mice for each group. One-way ANOVA with Dunnett’s test. c The postnatal survival of Nek1 Kat2J/Kat2J mice on control diet or ketogenic diet. N = 7 for each group. Unpaired two-tailed Student’s t-test. d , e Gr1 immunohistostaining (brown) of cerebral cortex and thalamus sections from the brains of Nek1 Kat2J/Kat2J or WT mice (3 weeks old) fed with ketogenic diet or control diet ( d ) and the numbers of Gr1 positive cells per sections were quantified ( e ). Bar = 20 µm. n = 3 sections per group. One-way ANOVA with Dunnett’s test. f – g Cerebromicrovessles were isolated from the brains of 3 Nek1 +/+ mice and 3 Nek1 Kat2J/Kat2J mice fed with control diet or ketogenic diet. The lysates of isolated cerebromicrovessles of each genotypes were analyzed by western blotting with indicated antibodies ( f ) and quantified using ImageJ ( g ). n = 3 replicates for each group. One-way ANOVA with Dunnett’s test. Uncropped blots in the Source Data file. h - i The mRNAs isolated from brains ( h ) and spinal cords ( i ) of Nek1 +/+ mice and Nek1 Kat2J/Kat2J mice fed with control diet or ketogenic diet (n=3 per group, 3 weeks old) were quantified for the levels of indicated cytokines by qPCR. Data are presented as mean ± SD. One-way ANOVA with Dunnett’s test.

Article Snippet: Antibodies against the following proteins were used for western blot analysis: NEK1 (Abcam #ab229489, 1:1000), NEK1 (Santa Cruz #sc-398813, 1:500), p-S166 RIPK1 (CST #31122, 1:1000), p-S166 RIPK1 (Arigo # ARG66476, 1:1000), RIPK1 (CST #3493, 1:2000), A20 (CST, #5630, 1:1000), TNFR1 (CST #13377, 1:1000), IκBα (CST #9242, 1:2000), p-S32/36 IκBα (CST, #9246, 1:2000), FLAG (Sigma-Aldrich #F3165, 1:2000), HA (CST #3724, 1:2000), α-synuclein (Invitrogen #AHB0261, 1:2000), p-S345 MLKL (CST #91689, 1:1000), MLKL (CST #37705, 1:1000), p-T231/S232 RIPK3 (CST #57220, 1:1000), RIPK3 (CST #13526, 1:1000), p-S79 acetyl-CoA carboxylase (CST #3661, 1:1000), acetyl-CoA carboxylase (CST #3662, 1:1000), SQSTM1/p62 (CST #5114, 1:5000), LAMP1 (Santa Cruz #sc-19992, 1:5000), p-Ser(EMD Millipore #05-1000, 1:2000).

Techniques: Western Blot, Control, Injection, Two Tailed Test, Isolation

Journal: Nature Communications

Article Title: NEK1-mediated retromer trafficking promotes blood–brain barrier integrity by regulating glucose metabolism and RIPK1 activation

doi: 10.1038/s41467-021-25157-7

Figure Lengend Snippet: Phosphorylation of S302/S304 VPS26B by NEK1 modulates the recycling of SNX27-retromer and SNX2-retromer that deliver proteins from endosomes to cell surface membrane and lysosomes. NEK1 deficiency blocks the recycling of SNX27-retromer from cell surface to endosomes. Since SNX27-retromer is important for delivering GLUT1 to the cell surface membrane, NEK1 deficiency leads to reduced GLUT1 levels at the cell surface membrane which reduces glucose uptake. Reduced glucose uptake disrupts mitochondrial metabolism and reduces cellular levels of acetyl-CoA. Reduced acetyl-CoA inhibits the acetylation of A20 which promotes its lysosomal degradation. Reduced levels of A20 sensitizes to the activation of RIPK1-dependent cell death and inflammation which can be inhibited by Nec-1s and also by ketogenic diet which provides alternate source of acetyl-CoA to restore cellular levels of A20.

Article Snippet: Antibodies against the following proteins were used for western blot analysis: NEK1 (Abcam #ab229489, 1:1000), NEK1 (Santa Cruz #sc-398813, 1:500), p-S166 RIPK1 (CST #31122, 1:1000), p-S166 RIPK1 (Arigo # ARG66476, 1:1000), RIPK1 (CST #3493, 1:2000), A20 (CST, #5630, 1:1000), TNFR1 (CST #13377, 1:1000), IκBα (CST #9242, 1:2000), p-S32/36 IκBα (CST, #9246, 1:2000), FLAG (Sigma-Aldrich #F3165, 1:2000), HA (CST #3724, 1:2000), α-synuclein (Invitrogen #AHB0261, 1:2000), p-S345 MLKL (CST #91689, 1:1000), MLKL (CST #37705, 1:1000), p-T231/S232 RIPK3 (CST #57220, 1:1000), RIPK3 (CST #13526, 1:1000), p-S79 acetyl-CoA carboxylase (CST #3661, 1:1000), acetyl-CoA carboxylase (CST #3662, 1:1000), SQSTM1/p62 (CST #5114, 1:5000), LAMP1 (Santa Cruz #sc-19992, 1:5000), p-Ser(EMD Millipore #05-1000, 1:2000).

Techniques: Phospho-proteomics, Membrane, Activation Assay