EMBO/SourceData huggingface dataset:
Figure 1 (B) SOFG staining of spinal entheseal tissue and Immunohistochemical analysis of CaSR+ and Runx2+ cells in human spinal tissue. The bottom panels show higher magnification of the boxed area in the top panels. Data information: SP: spinous process; IL: interspinous ligament; SL: supraspinous ligament; UIL: uncalcified interspinous ligament; TZ: transitional zone. Data shown as mean ± SD.. ** P < 0.01 compared between groups. Scale bar: 100μm  (C) Quantitative analysis of the number of CaSR+ and Runx2+ cells per area (mm2). n=5, Student's t-test.  D) RT-qPCR analysis of expression of CaSR in human spinal tissue. n=3, Student's t-test.  E) Western blot analysis of expression of CaSR in human spinal tissue.  F) Immunofluorescence analysis of CaSR+ (Green) and Runx2+ (Red) cells in human spinal tissue. (G) Quantitative analysis of CaSR+, Runx2+ and CaSR+ Runx2+ cells number per area (mm2) and CaSR+ cell percentage. n=5, Student's t-test. ata information: SP: spinous process; IL: interspinous ligament; SL: supraspinous ligament; UIL: uncalcified interspinous ligament; TZ: transitional zone. Data shown as mean ± SD.. ** P < 0.01 compared between groups. Scale bar: 100μm. H) Immunofluorescence analysis of CaSR+ (Green) and OCN+ (Red) cells in human spinal tissue. (I) Quantitative analysis of CaSR+, OCN+ and CaSR+ OCN+ cells number per area (mm2). n=5, Student's t-test. ata information: SP: spinous process; IL: interspinous ligament; SL: supraspinous ligament; UIL: uncalcified interspinous ligament; TZ: transitional zone. Data shown as mean ± SD.. ** P < 0.01 compared between groups. Scale bar: 100μm.
Figure 2 (A) μCT images of the spine in PGIS model (2D and 3D reconstruction). Arrow head shows spinal ankylosis. n=5 per group. μCT images of new bone (red area) in hind paws (2D and 3D reconstruction) in DBA/1 model compared to baseline.    (B) Incidence of spinal ankylosis in PGIS model. n=5 per cage of total 3 cages per group, Fisher's exact test. (C) Quantitative analysis of structural parameters of spinal ankylosis by μCT analysis. n=5 per group, one‐way ANOVA, Bonferroni post hoc. (D) RT-qPCR analysis of expression of CaSR in spinal ligament tissuse. n=3, Student's t-test. (E) H&E, SOFG and Immunofluorescence analyses of the spine specimen of PGIS model compare to baseline. n=5 per group. (F) Quantitative analysis of CaSR+, Runx2+ and CaSR+ Runx2+ cells number and CaSR+ cell percentage in the spine specimen of PGIS model. n=5 per group, one‐way ANOVA, Bonferroni post hoc. Data information: AF: AF: Annulus fibrosus; GP: Growth plate; NB: New bone. Data shown as mean ± SD. ** P < 0.01 compared between groups. Scale bar: 100μm. (G) H&E, SOFG and Immunofluorescence analyses of the spine specimen of PGIS model at 30 Weeks compare to baseline. GP: Growth plate. n=5 per group. (H) Quantitative analysis of CaSR+, OCN+ and CaSR+ OCN+ cells number in the spine specimen of PGIS model. n=5 per group, one‐way ANOVA, Bonferroni post hoc. Data information: AF: AF: Annulus fibrosus; GP: Growth plate; NB: New bone. Data shown as mean ± SD. ** P < 0.01 compared between groups. Scale bar: 100μm.
Figure 3 (A) μCT images of pathological new bone formation in DBA/1 model. n=9 per group.  (B) Incidence of ankle pathological new bone formation in DBA/1 model. n=9 per cage of total 3 cages per group, Fisher's exact test.  (C) Quantitative analysis of structural parameters of pathological new bone by μCT analysis. n=9 per group, one‐way ANOVA, Bonferroni post hoc.  (D) RT-qPCR analysis of expression of CaSR in hind paw tissue. n=3, Student's t-test.  (E) H&E, SOFG and Immunofluorescence analyses of pathological new bone formation in hind paws. n=9 per group. (F) Quantitative analysis of CaSR+, Runx2+ and CaSR+ Runx2+ cells number and CaSR+ cell percentage in pathological new bone formation sites. n=9 per group, one‐way ANOVA, Bonferroni post hoc. Data information: CB: Cortical bone; NB: New bone. Data shown as mean ± SD. ** P < 0.01 compared between groups. Scale bar: 100μm. G) H&E, SOFG and Immunofluorescence analyses of pathological new bone in hind paws. n=9 per group. (H) Quantitative analysis of CaSR+, OCN+ and CaSR+ OCN+ cells number in pathological new bone formation sites. n=9 per group, one‐way ANOVA, Bonferroni post hoc. Data information: CB: Cortical bone; NB: New bone. Data shown as mean ± SD. ** P < 0.01 compared between groups. Scale bar: 100μm.
Figure 4 (A) μCT images of the spine in PGIS model (2D and 3D reconstruction). Arrow head shows spinal ankylosis.  (B) H&E and SOFG staining of spine in PGIS model.  (C) Incidence of spinal ankylosis in PGIS model. n=5 per cage of total 6 cages per group, Fisher's exact test.  (D) Quantitative analysis of structural parameters of new bone by μCT analysis. n=5, one‐way ANOVA, Bonferroni post hoc.  (E) μCT images of new bone formation (red area) in hind paws (2D and 3D reconstruction).  (F) Incidence of new bone formation in DBA/1 model. n=9 per cage of total 3 cages per group, Fisher's exact test. (G) Quantitative analysis of structural parameters of new bone by μCT analysis. n=9, one‐way ANOVA, Bonferroni post hoc.  (H) H&E and SOFG staining of new bone in hind paws. Data information: CB: Cortical bone; NB: New bone  (I) μCT images of the PCT in SMTS model (2D). Arrow heads shows bony projection.  (J) H&E and SOFG staining of Achilles tendon enthesis compartment in SMTS model. Data information: UF: Uncalcified fibrocartilage; CF: Calcified fibrocartilage; PCT: posterior calcaneal tuberosity.  (K) Quantitative analysis of area of UF. n=5, one‐way ANOVA, Bonferroni post hoc. (L) Quantitative analysis of area of CF. n=5, one‐way ANOVA, Bonferroni post hoc. Data information: UF: Uncalcified fibrocartilage; CF: Calcified fibrocartilage
Figure 5 (A) Alizarin red staining of hBMSC cells for 11 days. (B) Quantification of Alizarin Red staining at 570 nm (A570). n=3, one‐way ANOVA, Bonferroni post hoc. (C) RT-qPCR analysis of osteogenesis markers in hBMSC cells. n=3, one‐way ANOVA, Bonferroni post hoc.  (D) Alizarin red staining of hBMSC cells. (E) Quantification of Alizarin Red staining at 570 nm (A570). n=3, one‐way ANOVA, Bonferroni post hoc.(F) RT-qPCR analysis of osteogenesis markers in hBMSC cells. n=3, one‐way ANOVA, Bonferroni post hoc. (G) Western blot analysis of phosphorylation of PLCγ protein levels in hBMSC cells.  (H) Western blot analysis of phosphorylation of PLCγ protein levels in hBMSC cells treated with SR and NPS-2143.  (I) Immunofluorescence analysis of spine in PGIS model. Quantitative analysis of CaSR+, p-PLCγ+ and CaSR+ p-PLCγ+ cells number in spine of PGIS model. n=5, one‐way ANOVA, Bonferroni post hoc. Data information: AF: Annulus fibrosus; NB: New bone  (J) Immunofluorescence analysis of Achilles tendon enthesis compartment in SMTS model. Quantitative analysis of CaSR+, p-PLCγ+ and CaSR+ p-PLCγ+ cells number in Achilles tendon enthesis compartment. n=5, one‐way ANOVA, Bonferroni post hoc. Data information: UF: Uncalcified fibrocartilage; CF: Calcified fibrocartilage  (K) Immunofluorescence analysis of pathological new bone on dorsal surface of hind paws. Quantitative analysis of CaSR+, p-PLCγ+ and CaSR+ p-PLCγ+ cells number in pathological new bone formation sites. n=9, one‐way ANOVA, Bonferroni post hoc. Data information: CB: Cortical bone; NB: New bone  (L) μCT images of the PCT in SMTS model (2D). Arrow heads shows bony projection.  (M) SOFG staining of Achilles tendon enthesis compartment in SMTS model. Data information: UF: Uncalcified fibrocartilage; CF: Calcified fibrocartilage; PCT: posterior calcaneal tuberosity. (N) Quantitative analysis of area of UF and CF. n=5, Student's t-test
Figure 6 (A) RT-qPCR and Western Blot analyses of CaSR expressions in MC3T3-E1 cells treated with IL-1β. n=3, one‐way ANOVA, Bonferroni post hoc.  (B) RT-qPCR and Western Blot analyss of CaSR expressions in MC3T3-E1 cells treated with TNFα. n=3, one‐way ANOVA, Bonferroni post hoc.  (C) RT-qPCR and Western Blot analyses of CaSR expressions in MC3T3-E1 cells treated with IL-17A. n=3, one‐way ANOVA, Bonferroni post hoc.  (D) RT-qPCR and Western Blot analyses of CaSR expressions in MC3T3-E1 cells treated with IL-22. n=3, one‐way ANOVA, Bonferroni post hoc.  (E) RT-qPCR and Western Blot analyses of CaSR expressions in MC3T3-E1 cells treated with IL-23. n=3, one‐way ANOVA, Bonferroni post hoc.
Figure 7 (A) Immunofluorescence analysis of spine in PGIS mice. Quantitative analysis of CaSR+ p-p65+ cells and CaSR+ p-Stat3+ cells number in pathological new bone formation of PGIS model. n = 5, one‐way ANOVA, Bonferroni post hoc. Data information: AF: Annulus fibrosus; GP: Growth plate; NB: New bone  (B) Immunofluorescence analysis of pathological new bone of hind paws in DBA/1 mice. Quantitative analysis of CaSR+ p-p65+ cells and CaSR+ p-Stat3+ cells number in pathological new bone formation sites. n = 9, one‐way ANOVA, Bonferroni post hoc. CB: Cortical bone; NB: New bone  (C) Immunofluorescence analysis of Achilles tendon enthesis compartment in SMTS model. Quantitative analysis of CaSR+ p-p65+ cells and CaSR+ p-stat3+ cells number in PCT. n=5, Student's t-test. UF: Uncalcified fibrocartilage; CF: Calcified fibrocartilage
Figure 2Three sex chromosome axes could be detected in the 2-month-old XUsp26+XUsp26-Y spermatocytes. Immunofluorescence analysis of SYCP3 (red) and SYCP1 (green) in (J), ATR (red) and SYCP3 (green) in (K) was performed in 2-month-old WT and XUsp26+XUsp26-Y spermatocytes. Nuclei were stained with DAPI (blue).The arrows indicate the Y chromosome. The arrowheads indicate the X chromosome.
Figure 3D Histological analysis of the seminiferous tubules and caudal epididymis of the 6-month-old Usp26+/Y and Usp26-/Y mice.F The deletion of Usp26 causes pachytene and meiotic division arrest. Representative PAS-hematoxylin staining in 6-month-old Usp26+/Y and Usp26-/Y seminiferous tubules. Parafﬁn sections from Usp26+/Y and Usp26-/Y testes were stained with PAS-hematoxylin. P: pachytene spermatocyte, D: diplotene spermatocyte, rST: round spermatid, eST: elongating spermatid, M: meiotic spermatocyte, aP: apoptotic pachytene spermatocytes, aM: abnormal meiotic divisions.
Figure 4E The X and Y chromosomes were unpaired in Usp26-/Y spermatocytes. Immunofluorescence analysis of Chr X-FISH (green), Chr Y-FISH (red), and SYCP3 (white) was performed in 6-month-old Usp26+/Y and Usp26-/Y spermatocytes. The arrowheads indicate the X chromosome. F Quantification of unpaired X and Y chromosomes in 6-month-old Usp26+/Y and Usp26-/Y mice (n= 5 independent experiments). Red dots indicate Usp26+/Y mice and green dots indicate Usp26-/Y mice.Sex chromosome recombination was perturbed in 6-month-old Usp26-/Y mice. Immunofluorescence analysis of SYCP3 (green), MRE11 (red) (G); was performed in 6-month-old Usp26+/Y and Usp26-/Y spermatocytes. Nuclei were stained with DAPI (blue). The arrows indicate the sex chromosomes.Sex chromosome recombination was perturbed in 6-month-old Usp26-/Y mice. Immunofluorescence analysis of ; SYCP3 (green), γH2AX (red) (H); SYCP3 (green), ATR (red), p-ATM (pink) (I) was performed in 6-month-old Usp26+/Y and Usp26-/Y spermatocytes. Nuclei were stained with DAPI (blue). The arrows indicate the sex chromosomes.L Crossover on sex chromosomes was impaired in 6-month-old Usp26-/Y mice. Immunofluorescence analysis of SYCP3 (green), MLH1 (red), and ATR (white) was performed in 6-month-old Usp26+/Y and Usp26-/Y spermatocytes. Nuclei were stained with DAPI (blue).
Figure 5D Representative TUNEL results in Usp26+/Y and Usp26-/Y testes. Parafﬁn sections from 6-month-old Usp26+/Y and Usp26-/Y testes were stained with TUNEL (green) and DAPI (blue) to show dead cells in stage IV, VIII and XII tubules with pachytene and metaphase spermatocytes, respectively. The arrowheads indicate the laggard chromosomes. P: pachytene spermatocytes, rSt: round spermatid, spz: spermatozoa, M: meiotic spermatocyte, aP: apoptotic pachytene spermatocytes, aM: abnormal meiotic divisions, arSt: apoptotic round spermatid.E The protein level of three known SAC proteins was reduced in 2-month-old and 6-month-old Usp26−/Y mouse testes. Immunoblotting of MAD2, BUBR1, PLK1 and USP26 was performed in 2-month-old and 6-month-old Usp26+/Y and Usp26-/Y testes. H3Ser10p and Tub served as loading control.F Usp26-deficient mice produced XY aneuploid round spermatids. FISH analysis of Chr X (green) and Chr Y (red) was performed in 2-month-old and 6-month-old Usp26+/Y and Usp26−/Y round spermatids. Nuclei were stained with DAPI (blue). The arrows indicate the Y chromosome, and the arrowheads indicate the X chromosome. G Quantification of different types of round spermatids in 2-month-old Usp26 +/Y, Usp26-/Y mice (n= 3 independent experiments) and 6-month-old Usp26+/Y (n= 3 independent experiments), Usp26-/Y mice (n= 4 independent experiments). P=0.0026 for XY spermatozoa in 6-month-old Usp26+/Y and Usp26−/Y mice. P=0.0216 for O spermatozoa in 6-month-old Usp26+/Y and Usp26−/Y mice. H Usp26-deficient mice produced XY aneuploid spermatozoa. FISH assay of Chr X (green) and Chr Y (red) was performed in 2-month-old and 6-month-old Usp26+/Y and Usp26−/Y spermatozoa. Nuclei were stained with DAPI (blue). The arrows indicate the Y chromosome, and the arrowheads indicate the X chromosome. I Quantification of different types of spermatozoa in 2-month-old Usp26+/Y, Usp26-/Y mice (n= 3 independent experiments) and 6-month-old Usp26+/Y, Usp26-/Y mice (n= 5 independent ex experiments). P=0.0195 for XY spermatozoa in 6-month-old Usp26+/Y and Usp26−/Y mice. Data are presented as means ± SD.
Figure 6The X and Y chromosomes were unpaired in Tex11-/Y spermatocytes. Immunofluorescence analysis of SYCP3 (green), TRF1 (red), ATR (white) (D); SYCP3 (green), MLH1 (red), ATR (white) (E) was performed in in Tex11+/Y and Tex11-/Y spermatocytes. Nuclei were stained with DAPI (blue). The arrowheads indicate the X chromosome.
Figure 7A, B Quantification of different types of spermatozoa in fertile men with or without USP26 mutations. The spermatozoa from fertile men without USP26 mutations (n = 10 independent experiments); fertile men contained the USP26 c.463C>T mutation (The sample was analyzed in three repeated experiments), the USP26 c.125T>C mutation (n = 2 independent experiments, one sample was analyzed in two repeated experiments), the USP26 c.1044T>A mutation (n = 5 independent experiments), and the USP26 c.370_371insACA/494T>C/1423C>T mutation (n = 10 independent experiments) were collected to perform Chr X and Chr Y FISH. P-values are shown for comparison to fertile men without USP26 mutations.C The fertile men with the USP26 mutated haplotype produced XY and O aneuploid spermatozoa. FISH assay of Chr X (green) and Chr Y (red) was performed in fertile men with or without the USP26 mutated haplotype. Nuclei were stained with DAPI (blue). The arrows indicate the Y chromosome, and the arrowheads indicate the X chromosome.
Figure 1Survival rates of control and Cdc50a cKO mice after oil or 4-OHT injection (n = 5 for each group, P*** = 0.0003).Bar graphs showing audiogenic seizure scores following auditory stimuli (5, 10, 15, 20, 40 kHz or white noise (WN) at 70, 80, 90, or 100 dB) in control (E) and Cdc50a cKO (F) mice (n = 8 for each group).Representative confocal z-stack images showing cFos (magenta)-positive neurons from control and Cdc50a cKO mice (CTX: cortex, HP: hippocampus, and IC: inferior colliculus). Scale bar, 20 μm. Bar graphs showing the relative area of cFos-positive cells in the brains of control and Cdc50a cKO mice (n = 5 for each group, **P = 0.0030, n.s., not significant).
Figure 2Schematic diagram of the experiment. Representative confocal z-stack images of PSD95 (green)- and VGLUT1 (red)-positive excitatory (A) and Gephyrin (green)- and VGAT (magenta)-positive inhibitory (B) synapses in the CTX, HP, and IC (CTX: cortex, HP: hippocampus, and IC: inferior colliculus) of control and Cdc50a cKO mice. The dashed line represents where the high magnified insert was taken from. Scale bar, 20 μm.Representative current clamp traces from the IC of control and Cdc50a cKO mice; +100 pA (upper) and +400 pA (lower) currents were injected into control mice (left) and Cdc50a cKO mice (right) for 500 ms.
Figure 3The bar graphs show the relative percentages of neurons showing PS exposure only in the soma (left) and overall PS exposure (right) (n = 30-40 cells for each group) in control and Cdc50a cKO mice.Schematic diagram of imaging of the neuronal somas and proximal and distal dendrites of neurons in the CA1 region of the HP. Representative confocal z-stack images showing Gephyrin-positive inhibitory post-synapses in the somas (red) and proximal (green) and distal dendrites (cyan) of neurons in the CA1 region of the HP in control and Cdc50a cKO mice. Scale bar, 20 μm. Bar graphs showing the number of Gephyrin-positive inhibitory post-synapses in the somas and proximal and distal dendrites of neurons in the CA1 of the HP in control and Cdc50a cKO mice (n = 4 for each group, P*** = 0.0002, P**** < 0.0001).
Figure 4Representative confocal z-stack and colocalization images of Gephyrin (green), IBA1 (blue), and CD68 (red) in the IC of control and Cdc50a cKO mice. Scale bar, 20 μm. Bar graphs showing the relative colocalization area of Gephyrin and CD68 in the IC of control and Cdc50a cKO mice (n = 3 for each group, **P = 0.0024).Representative confocal z-stack images of microglia (IBA1, green, upper) and inhibitory synapses (Gephyrin: green, VGAT: red, lower) in the IC of control;DMSO, control;PLX3397, Cdc50a cKO;DMSO and Cdc50a cKO;PLX3397 mice. The dashed line represents where the high magnified insert was taken from. Scale bar, 20 μm. Bar graphs showing the relative area of IBA1-positive cells in the IC of control;DMSO, control;PLX3397, Cdc50a cKO;DMSO and Cdc50a cKO;PLX3397 mice (n = 3 for each group, control;DMSO versus control;PLX3397, *P = 0.0428, control;DMSO versus Cdc50a cKO;DMSO, ***P = 0.0002, Cdc50a cKO;DMSO versus Cdc50a cKO;PLX3397, ***P = 0.0005). Bar graphs showing the number of inhibitory (Gephyrin- and VGAT-positive) synapses in the IC of control;DMSO, control;PLX3397, Cdc50a cKO;DMSO and Cdc50a cKO;PLX3397 mice (n = 3 for each group, P**** < 0.0001).
Figure 5Representative confocal z-stack images of Gephyrin (green)- and VGAT (red)-positive inhibitory synapses in the IC (inferior colliculus) of control;C3+/+, control;C3-/-, Cdc50a cKO;C3+/+ and Cdc50a cKO;C3-/- mice. The dashed line represents where the high magnified insert was taken from. Scale bar, 10 μm.Representative confocal z-stack images of Gephyrin (green)- and VGAT (red)-positive inhibitory synapses in the IC of control;Mertk+/+, control;Mertk-/-, Cdc50a cKO;Mertk+/+ and Cdc50a cKO;Mertk-/- mice. The dashed line represents where the high magnified insert was taken from. Scale bar, 20 μm.
Figure 6Schematic diagram of the experiment in which PS exposure was measured in the brains of 1-month- and 3-month-old wild-type mice by injecting AAV9-GFAP-secA5-mCherry in the neonatal stage (P0). Representative confocal z-stack colocalization images of Gephyrin (green), VGAT (blue), and secA5 (red) in the HP (hippocampus) of 1-month- and 3-month-old wild-type mice. The white circles indicate secA5-positive inhibitory synapses. Scale bar, 10 μm. Bar graphs showing the expression of secA5 (B) and IBA1 (C) in inhibitory post-synapses (Gephyrin-positive, green) and pre-synapses (VGAT-positive, blue) in the HP of 1-month- and 3-month-old wild-type mice (n = 4 for each group, ***P = 0.0009, *P = 0.0248, n.s., not significant).Schematic diagram of the experiment in which Annexin V was injected intravenously (every two days) to block PS in the brains of 1-month- and 3-month-old wild-type mice. Representative confocal z-stack images of Gephyrin (green)- and VGAT (red)-positive inhibitory synapses in the HP of 1-month-old wild-type mice. The white circles indicate inhibitory synapses. Scale bar, 10 μm. Bar graphs showing the number of inhibitory (Gephyrin- and VGAT-positive) synapses in the HP of 1-month-old mice after PBS or Annexin V injection (n = 4 for each group, ***P = 0.0013, **P = 0.0090).Schematic diagram of the experiment in which microglial Mertk KO (CX3CR1-CreERT2;Mertkfl/fl) and control (Mertkfl/fl) mice were generated. Both 1-month-old microglial Mertk KO mice and 1-month-old control mice were injected with 4-OHT for 5 consecutive days. Representative confocal z-stack images of Gephyrin (green)- and VGAT (magenta)-positive inhibitory synapses in the HP of Mertkfl/fl and CX3CR1-CreERT2;Mertkfl/fl mice. The white circles indicate inhibitory synapses. Scale bar, 10 μm. Bar graphs showing the number of inhibitory (Gephyrin- and VGAT-positive) synapses in the HP of Mertkfl/fl and CX3CR1-CreERT2;Mertkfl/fl mice (n = 4 for each group, **P = 0.0027, *P = 0.0062). Representative confocal z-stack colocalization images of Gephyrin (green), VGAT (blue) and secA5 (red) in the HP of Mertkfl/fl and CX3CR1-CreERT2;Mertkfl/fl mice. The white circles indicate secA5-positive inhibitory synapses. Scale bar, 10 μm. Bar graphs showing the relative expression of secA5 in inhibitory synapses in the HP of Mertkfl/fl and CX3CR1-CreERT2;Mertkfl/fl mice (n = 4 for each group, *P = 0.0015).
Figure 1(A) 293T cells were transfected with Flag-ULK1 or control empty vector and subjected to immunoprecipitation with anti-Flag antibody, followed by immunoblot analysis with the indicated antibodies.(B) 293T cells were co-transfected with Flag-AMPKα2 and HA-ULK1. Cell lysates were subjected to immunoprecipitation using anti-Flag antibody followed by SDS-PAGE/immunoblot analysis with anti-HA and anti-Flag antibodies.(C) 293T cells were transiently transfected with Flag-AMPKα2 or Flag-AMPKβ1. After 24 h, immunoprecipitation was performed using anti-Flag or control anti-HA antibodies and analyzed by immunoblotting with anti-ULK1 and anti-Flag polyclonal antibodies.(D) 293T cell lysates were subjected to immunoprecipitation with anti-ULK1 polyclonal antibody or control preimmune rabbit serum (NRS), followed by immunoblot analysis with anti-ULK1 and anti-AMPKα antibodies.(E) 293T cells were infected with ULK1 shRNA or control shRNA lentiviruses and subjected to immunoprecipitation with control rabbit IgG or anti-AMPKα antibody followed by immunoblotting with anti-ULK1 and anti-AMPKα antibodies.(F) Purified His6-AMPKα1/β1/γ1 fusion proteins (250 ng) were mixed with purified His6-tagged ULK1 (1 µg) or Bcl-XL (200 ng) proteins in 1% Triton X-100 lysis buffer containing protease inhibitors and subjected to immunoprecipitation with anti-ULK1 antibody. The resulting protein complexes and 10% of the input proteins were analyzed by immunoblotting with anti-His-Tag polyclonal antibody.
Figure 2(B, C) 293T cells were transfected with empty vector or HA-tagged wild type (WT) or deletion mutants of ULK1 and subjected to immunoprecipitation with anti-HA antibody. The resulting immune complexes were analyzed by immunoblotting with antibodies specific for mTOR, raptor, AMPKα or HA-tag.