Journal: Scientific Reports

Article Title: Furin-mediated cleavage of LRP1 and increase in ICD of LRP1 after cerebral ischemia and after exposure of cultured neurons to NMDA

doi: 10.1038/s41598-019-48279-x

Figure Lengend Snippet: ( a ) Schematic drawing of brain regions used in the experiments. ( b ) Changes in α-chain, β-chain, and ICD of LRP1 at 24 h after MCAO/R. Proteins from non-ischemic (N) and ischemic (I) areas from the ipsilateral (ipsi) and contralateral (cont) cortex in MCAO/R rats were analyzed by Western blotting with anti-α-chain and anti-β-chain/ICD of LRP1, and anti-β-actin antibodies. Cropped blots are displayed and full-length blots are presented in Supplementary Fig. . Bands corresponding to α-chain ( c ), β-chain ( d ), and ICD ( e ) of LRP1 and β-actin were scanned, and the scanned bands were normalized by the untreated naïve control on the same blot. β-Actin was used as a loading control. Results are the means ± SD (n = 3 rats per group). *Indicates a significant difference from the corresponding area of the contralateral hemisphere (p < 0.05). ( f ) Changes in localization of β-chain and/or ICD of LRP1 and TGN at 24 h after MCAO/R. Non-ischemic and ischemic areas of the ipsilateral and contralateral hemispheres in the brain of MCAO/R rats were immunostained with anti-TGN46 and anti-β-chain/ICD of LRP1 antibodies, and then observed with a confocal microscope. The colocalization of TGN46 with LRP1-ICD is indicated by the “white arrow”. Representative images are shown from one rat. The scale bar represents 30 µm. ( g ) Changes in localization of β-chain and/or ICD of LRP1 and TGN at 24 h after MCAO/R. The number of LRP1-ICD immune-positive cells that co-localized with TGN46 was counted. Results are expressed as the means ± SD (n = 4 rats per group). *Indicates a significant difference from the corresponding area of the contralateral hemisphere (P < 0.05). The range of the number of cells counted for N-cont, I-cont, N-ipsi, and I-ipsi were 22–60/rat (the total number of cells counted: 150), 14–66/rat (total: 139), 22–59/rat (total: 159), and 23–63/rat (total: 156), respectively. The total number of cells counted in Fig. 1g was 604.

Article Snippet: For immunocytochemistry, neurons (10 DIV) on glass coverslips pre-coated with poly-D-lysine were washed with PBS (300 μl) twice for 5 min each time and fixed with 4% paraformaldehyde at room temperature for 10 min. After having been washed twice with PBS (300 μl) for 5 min each time, the neurons were made permeable with 0.2% TritonX-100 in PBS (300 μl) by incubation at room temperature for 10 min and then blocked with a mixture of 10% normal goat serum, 1% BSA, and 0.2% Triton X-100 in PBS (300 μl) at room temperature for 1 h. The neurons were incubated with rabbit anti-LRP1 (dilution, 1:2000; 2703-1, Epitomics), goat anti-LRP1 [C II] (dilution, 1:100; AF2368, R&D), mouse anti-TGN46 (dilution 1:200, ab2809, Abcam) or mouse anti-furin (dilution 1:500; sc-133141, Santa cruz) in PBS containing a mixture of 10% normal goat or donkey serum, 1% BSA, and 0.2% Triton X-100 (300 μl) at room temperature for 1 h. The cells were washed 3 times with PBS (300 μl) for 5 min, and then incubated with Alexa Fluor 488-conjugated donkey anti-rabbit IgG (dilution 1:200, Thermo Fisher) and Alexa Fluor 594-conjugated goat anti-mouse IgG (dilution 1:200, Thermo Fisher), and donkey anti-goat IgG at room temperature for 1 h. Subsequently, the neurons were washed 3 times, 5 min each time, with 300 μl of PBS containing Hoechst33342 (0.5 μg/ml, 346-07951, Dojindo, Kumamoto, Japan) and mounted with Fluoromount/Plus (DiagnosticBioSystems, Pleasanton,CA).

Techniques: Western Blot, Microscopy

Journal: Scientific Reports

Article Title: Furin-mediated cleavage of LRP1 and increase in ICD of LRP1 after cerebral ischemia and after exposure of cultured neurons to NMDA

doi: 10.1038/s41598-019-48279-x

Figure Lengend Snippet: Effects of NMDA treatment on protein levels of α-chain, β-chain, and ICD of LRP1 at 4 h after 0 µM (0) or 30 µM (30) NMDA treatment. Proteins from cortical neurons in primary culture were analyzed by Western blotting with anti-α-chain and anti-β-chain/ICD of LRP1, and anti-β-actin antibodies ( a ). Cropped blots are displayed and full-length blots are presented in Supplementary Fig. . Bands corresponding to the α-chain ( b ), β-chain ( c ), and ICD ( d ) of LRP1 and β-actin were scanned, and the scanned bands were normalized by reference to the untreated control on the same blot. β-Actin was used as a loading control. Results are the means ± SD (n = 3 independent experiments). *Indicates a significant difference from the NMDA-untreated group (p < 0.05). ( e ) Effects of NMDA treatment on localization of α-chain and β-chain/ICD at 4 h after 0 µM (0) or 30 µM NMDA (30) treatment. Cortical neurons in primary culture were treated with NMDA were immunostained with anti-α-chain (red) and anti-β-chain/ICD (green), which recognizes ICD domain, of LRP1 antibodies. Representative images are shown from one experiment. The scale bar represents 30 µm. ( f ) The number of cells where LRP1-ICD was localized in the perinuclear region was counted. Results are expressed as the means ± SD of 4 independent experiments. *Significant difference from the NMDA-untreated group (NMDA [0]) (P < 0.05). The range of the number of cells counted for NMDA [0] and NMDA [30] were 28–42/experiment (the total number of cells counted: 138) and 32–87/experiment (total: 144), respectively. The total number of cells counted in Fig. 3f was 282.

Article Snippet: For immunocytochemistry, neurons (10 DIV) on glass coverslips pre-coated with poly-D-lysine were washed with PBS (300 μl) twice for 5 min each time and fixed with 4% paraformaldehyde at room temperature for 10 min. After having been washed twice with PBS (300 μl) for 5 min each time, the neurons were made permeable with 0.2% TritonX-100 in PBS (300 μl) by incubation at room temperature for 10 min and then blocked with a mixture of 10% normal goat serum, 1% BSA, and 0.2% Triton X-100 in PBS (300 μl) at room temperature for 1 h. The neurons were incubated with rabbit anti-LRP1 (dilution, 1:2000; 2703-1, Epitomics), goat anti-LRP1 [C II] (dilution, 1:100; AF2368, R&D), mouse anti-TGN46 (dilution 1:200, ab2809, Abcam) or mouse anti-furin (dilution 1:500; sc-133141, Santa cruz) in PBS containing a mixture of 10% normal goat or donkey serum, 1% BSA, and 0.2% Triton X-100 (300 μl) at room temperature for 1 h. The cells were washed 3 times with PBS (300 μl) for 5 min, and then incubated with Alexa Fluor 488-conjugated donkey anti-rabbit IgG (dilution 1:200, Thermo Fisher) and Alexa Fluor 594-conjugated goat anti-mouse IgG (dilution 1:200, Thermo Fisher), and donkey anti-goat IgG at room temperature for 1 h. Subsequently, the neurons were washed 3 times, 5 min each time, with 300 μl of PBS containing Hoechst33342 (0.5 μg/ml, 346-07951, Dojindo, Kumamoto, Japan) and mounted with Fluoromount/Plus (DiagnosticBioSystems, Pleasanton,CA).

Techniques: Western Blot

Journal: Scientific Reports

Article Title: Furin-mediated cleavage of LRP1 and increase in ICD of LRP1 after cerebral ischemia and after exposure of cultured neurons to NMDA

doi: 10.1038/s41598-019-48279-x

Figure Lengend Snippet: ( a ) Effects of NMDA treatment on the levels of α-chain, β-chain, and ICD of LRP1 at 0, 0.5, 1, 2, and 4 h after 0 µM (0) or 30 µM (30) NMDA treatment. Proteins from cortical neurons in primary culture were analyzed by Western blotting with anti-α-chain and anti-β-chain of LRP1, and anti-β-actin antibodies. Cropped blots are displayed and full-length blots are presented in Supplementary Fig. . ( b ) Bands corresponding to ICD of LRP1 and β-actin were scanned, and the scanned bands were normalized by reference to the untreated control on the same blot. β-Actin was used as a loading control. Results are the means ± SD (n = 3 independent experiments). *Indicates a significant difference from the NMDA-treated group at 0 h (p < 0.05). ( c ) Effects of NMDA treatment on localization of α-chain and β-chain/ICD of LRP1 at 0.5, 1, and 4 h after 0 µM (NMDA [0]) or 30 µM NMDA (NMDA [30])) treatment. Representative images are shown from one experiment. The scale bar represents 30 µm. ( d ) The number of cells where LRP1-ICD was localized in the perinuclear region was counted. Results are expressed as the means ± SD of 4 independent experiments. *Significant difference from the NMDA-treated group at 0.5 h (NMDA [30]) (P < 0.05). The range of the number of cells counted in NMDA [0]-0.5 h, NMDA [0]-1 h, NMDA [0]-4 h, NMDA [30]-0.5 h, NMDA [30]-1 h, and NMDA [30]-4 h, were 20–32/experiment (the total number of cells counted: 105), 30–57/experiment (total: 172), 18–35/experiment (total: 101), 27–74/experiment (total: 195), 19–40/experiment (total: 113), and 31–57/experiment (total: 174), respectively. The total number of cells counted in Fig. 4d was 860.

Article Snippet: For immunocytochemistry, neurons (10 DIV) on glass coverslips pre-coated with poly-D-lysine were washed with PBS (300 μl) twice for 5 min each time and fixed with 4% paraformaldehyde at room temperature for 10 min. After having been washed twice with PBS (300 μl) for 5 min each time, the neurons were made permeable with 0.2% TritonX-100 in PBS (300 μl) by incubation at room temperature for 10 min and then blocked with a mixture of 10% normal goat serum, 1% BSA, and 0.2% Triton X-100 in PBS (300 μl) at room temperature for 1 h. The neurons were incubated with rabbit anti-LRP1 (dilution, 1:2000; 2703-1, Epitomics), goat anti-LRP1 [C II] (dilution, 1:100; AF2368, R&D), mouse anti-TGN46 (dilution 1:200, ab2809, Abcam) or mouse anti-furin (dilution 1:500; sc-133141, Santa cruz) in PBS containing a mixture of 10% normal goat or donkey serum, 1% BSA, and 0.2% Triton X-100 (300 μl) at room temperature for 1 h. The cells were washed 3 times with PBS (300 μl) for 5 min, and then incubated with Alexa Fluor 488-conjugated donkey anti-rabbit IgG (dilution 1:200, Thermo Fisher) and Alexa Fluor 594-conjugated goat anti-mouse IgG (dilution 1:200, Thermo Fisher), and donkey anti-goat IgG at room temperature for 1 h. Subsequently, the neurons were washed 3 times, 5 min each time, with 300 μl of PBS containing Hoechst33342 (0.5 μg/ml, 346-07951, Dojindo, Kumamoto, Japan) and mounted with Fluoromount/Plus (DiagnosticBioSystems, Pleasanton,CA).

Techniques: Western Blot

Journal: Scientific Reports

Article Title: Furin-mediated cleavage of LRP1 and increase in ICD of LRP1 after cerebral ischemia and after exposure of cultured neurons to NMDA

doi: 10.1038/s41598-019-48279-x

Figure Lengend Snippet: ( a ) Effects of glutamate treatment on protein levels of β-chain and ICD of LRP1 at 4 h after 0 µM (0) or 30 µM (Glu) glutamate treatment of cortical neurons in primary cultures. Proteins from the cultures were analyzed by Western blotting with anti-β-chain/ICD of LRP1 and anti-β-actin antibodies. Representative blots of β-chain and ICD of LRP1 are shown ( a ). Cropped blots are displayed and full-length blots are presented in Supplementary Fig. . Bands corresponding to ICD of LRP1 and β-actin were scanned, and the scanned bands were normalized by referring to the untreated control on the same blot ( b ). β-Acitn was used as a loading control. Results are the means ± SD (Cortical neuron n = 8 independent experiments). *Indicates a significant difference between 0 µM and 30 µM glutamate treatments (p < 0.05). ( c ) Effect of NMDA treatment on cell viability of cortical neurons. Cortical neurons were incubated with Glia-derived ApoE-containing lipoproteins (LP) and/or 30 µM NMDA for 15 min. The relative cell viability was expressed as the percentage of the absorbance at 450 nm of each treatment group against that of the untreated control group. Results are the means ± SD (n = 4 independent experiments). *Indicates a significant difference from the NMDA- and LP-untreated control group (Con; p < 0.05).

Article Snippet: For immunocytochemistry, neurons (10 DIV) on glass coverslips pre-coated with poly-D-lysine were washed with PBS (300 μl) twice for 5 min each time and fixed with 4% paraformaldehyde at room temperature for 10 min. After having been washed twice with PBS (300 μl) for 5 min each time, the neurons were made permeable with 0.2% TritonX-100 in PBS (300 μl) by incubation at room temperature for 10 min and then blocked with a mixture of 10% normal goat serum, 1% BSA, and 0.2% Triton X-100 in PBS (300 μl) at room temperature for 1 h. The neurons were incubated with rabbit anti-LRP1 (dilution, 1:2000; 2703-1, Epitomics), goat anti-LRP1 [C II] (dilution, 1:100; AF2368, R&D), mouse anti-TGN46 (dilution 1:200, ab2809, Abcam) or mouse anti-furin (dilution 1:500; sc-133141, Santa cruz) in PBS containing a mixture of 10% normal goat or donkey serum, 1% BSA, and 0.2% Triton X-100 (300 μl) at room temperature for 1 h. The cells were washed 3 times with PBS (300 μl) for 5 min, and then incubated with Alexa Fluor 488-conjugated donkey anti-rabbit IgG (dilution 1:200, Thermo Fisher) and Alexa Fluor 594-conjugated goat anti-mouse IgG (dilution 1:200, Thermo Fisher), and donkey anti-goat IgG at room temperature for 1 h. Subsequently, the neurons were washed 3 times, 5 min each time, with 300 μl of PBS containing Hoechst33342 (0.5 μg/ml, 346-07951, Dojindo, Kumamoto, Japan) and mounted with Fluoromount/Plus (DiagnosticBioSystems, Pleasanton,CA).

Techniques: Western Blot, Incubation, Derivative Assay

Journal: Scientific Reports

Article Title: Furin-mediated cleavage of LRP1 and increase in ICD of LRP1 after cerebral ischemia and after exposure of cultured neurons to NMDA

doi: 10.1038/s41598-019-48279-x

Figure Lengend Snippet: ( a ) Effects of calpain inhibitor (calpeptin) on the levels of α-chain, β-chain and ICD of LRP1 at 4 h after 0 µM (0) or 30 µM (30) NMDA treatment with (+) or without (−) 30 µM calpain inhibitor. Proteins from cortical neurons in primary culture were analyzed by Western blotting with anti-α-chain and anti-β-chain/ICD of LRP1, and anti-β-actin antibodies. Cropped blots are displayed and full-length blots are presented in Supplementary Fig. . Bands corresponding to α-chain ( b ), β-chain ( c ), and ICD ( d ) of LRP1 and β-actin were scanned, and the scanned bands were normalized by reference to the untreated control on the same blot. β-Actin was used as a loading control. Results are the means ± SD (n = 4 independent experiments).

Article Snippet: For immunocytochemistry, neurons (10 DIV) on glass coverslips pre-coated with poly-D-lysine were washed with PBS (300 μl) twice for 5 min each time and fixed with 4% paraformaldehyde at room temperature for 10 min. After having been washed twice with PBS (300 μl) for 5 min each time, the neurons were made permeable with 0.2% TritonX-100 in PBS (300 μl) by incubation at room temperature for 10 min and then blocked with a mixture of 10% normal goat serum, 1% BSA, and 0.2% Triton X-100 in PBS (300 μl) at room temperature for 1 h. The neurons were incubated with rabbit anti-LRP1 (dilution, 1:2000; 2703-1, Epitomics), goat anti-LRP1 [C II] (dilution, 1:100; AF2368, R&D), mouse anti-TGN46 (dilution 1:200, ab2809, Abcam) or mouse anti-furin (dilution 1:500; sc-133141, Santa cruz) in PBS containing a mixture of 10% normal goat or donkey serum, 1% BSA, and 0.2% Triton X-100 (300 μl) at room temperature for 1 h. The cells were washed 3 times with PBS (300 μl) for 5 min, and then incubated with Alexa Fluor 488-conjugated donkey anti-rabbit IgG (dilution 1:200, Thermo Fisher) and Alexa Fluor 594-conjugated goat anti-mouse IgG (dilution 1:200, Thermo Fisher), and donkey anti-goat IgG at room temperature for 1 h. Subsequently, the neurons were washed 3 times, 5 min each time, with 300 μl of PBS containing Hoechst33342 (0.5 μg/ml, 346-07951, Dojindo, Kumamoto, Japan) and mounted with Fluoromount/Plus (DiagnosticBioSystems, Pleasanton,CA).

Techniques: Western Blot

Journal: Scientific Reports

Article Title: Furin-mediated cleavage of LRP1 and increase in ICD of LRP1 after cerebral ischemia and after exposure of cultured neurons to NMDA

doi: 10.1038/s41598-019-48279-x

Figure Lengend Snippet: ( a ) Effects of furin inhibitor on the levels of α-chain, β-chain, and ICD of LRP1 at 4 h after 0 µM (0) or 30 µM (30) NMDA treatment with (+) or without (−) 10 µM furin inhibitor. Proteins from cortical neurons in primary culture were analyzed by Western blotting with anti-α-chain and anti-β-chain of LRP1/ICD, and anti-β-actin antibodies. Cropped blots are displayed and full-length blots are presented in Supplementary Fig. . Bands corresponding to α-chain ( b ), β-chain ( c ), and ICD ( d ) of LRP1 and β-actin were scanned, after which the scanned bands were normalized by referring to the untreated control on the same blot. β-Actin was used as a loading control. Results are the means ± SD (n = 3 independent experiments). *Indicates a significant difference between 0 µM and 30 µM NMDA treatments without furin inhibitor (p < 0.05). ( e ) Effect of furin inhibitor on localization of α-chain (red) and β-chain of LRP1/ICD (green) at 4 h after 0 µM (control) or 30 µM NMDA (NMDA) treatment with or without 10 µM furin inhibitor. Representative images are shown from one experiment. The scale bar represents 30 µm. ( f ) The number of cells where LRP1-ICD was localized in the perinuclear region was counted. Results are expressed as the means ± SD of 4 independent experiments. *Significant difference from the NMDA- and furin inhibitor-untreated group (NMDA [0] and furin inhibitor [−]) (P < 0.05). # Significant difference from the NMDA-treated and furin inhibitor-untreated group (NMDA [30] and furin inhibitor [−]) (P < 0.05). The range of the number of counted cells in NMDA [0]-furin inhibitor [−], NMDA [0]-furin inhibitor [+], NMDA [30]-furin inhibitor [−], and NMDA [30]-furin inhibitor [+], were 14–36/experiment (the total number of cells counted: 110), 24–38/experiment (total: 128), 38–73/experiment (total: 216), and 27–57/experiment (total: 151), respectively. The total number of cells counted in Fig. 7f was 605.

Article Snippet: For immunocytochemistry, neurons (10 DIV) on glass coverslips pre-coated with poly-D-lysine were washed with PBS (300 μl) twice for 5 min each time and fixed with 4% paraformaldehyde at room temperature for 10 min. After having been washed twice with PBS (300 μl) for 5 min each time, the neurons were made permeable with 0.2% TritonX-100 in PBS (300 μl) by incubation at room temperature for 10 min and then blocked with a mixture of 10% normal goat serum, 1% BSA, and 0.2% Triton X-100 in PBS (300 μl) at room temperature for 1 h. The neurons were incubated with rabbit anti-LRP1 (dilution, 1:2000; 2703-1, Epitomics), goat anti-LRP1 [C II] (dilution, 1:100; AF2368, R&D), mouse anti-TGN46 (dilution 1:200, ab2809, Abcam) or mouse anti-furin (dilution 1:500; sc-133141, Santa cruz) in PBS containing a mixture of 10% normal goat or donkey serum, 1% BSA, and 0.2% Triton X-100 (300 μl) at room temperature for 1 h. The cells were washed 3 times with PBS (300 μl) for 5 min, and then incubated with Alexa Fluor 488-conjugated donkey anti-rabbit IgG (dilution 1:200, Thermo Fisher) and Alexa Fluor 594-conjugated goat anti-mouse IgG (dilution 1:200, Thermo Fisher), and donkey anti-goat IgG at room temperature for 1 h. Subsequently, the neurons were washed 3 times, 5 min each time, with 300 μl of PBS containing Hoechst33342 (0.5 μg/ml, 346-07951, Dojindo, Kumamoto, Japan) and mounted with Fluoromount/Plus (DiagnosticBioSystems, Pleasanton,CA).

Techniques: Western Blot

Journal: Scientific Reports

Article Title: Furin-mediated cleavage of LRP1 and increase in ICD of LRP1 after cerebral ischemia and after exposure of cultured neurons to NMDA

doi: 10.1038/s41598-019-48279-x

Figure Lengend Snippet: ( a ) Effect of furin inhibitor on localization of TGN46 and LRP1-ICD at 4 h after 0 µM (control) or 30 µM NMDA (NMDA) treatment of cortical neurons with or without 10 µM furin inhibitor. The cells were immunostained with anti-TGN46 and anti-β-chain/ICD antibodies. Representative images are shown from one experiment. The scale bar represents 30 µm. ( b ) The number of LRP1-ICD immune-positive cells that co-localized with TGN46 in the perinuclear region was counted. Results are expressed as the means ± SD of 4 independent experiments. *Significant difference from the NMDA- and furin inhibitor-untreated group (NMDA [0] and furin inhibitor [−]) (P < 0.05). # Significant difference from the NMDA-treated and furin inhibitor-untreated group (NMDA [30] and furin inhibitor [−]) (P < 0.05). The range of the number of counted cells in NMDA [0]-furin inhibitor [−], NMDA [0]-furin inhibitor [+], NMDA [30]-furin inhibitor [−], and NMDA [30]-furin inhibitor [+], were 29–40/experiment (the total number of cells counted: 133), 22–27/experiment (total: 98), 51–82/experiment (total: 186), and 20–32/experiment (total: 109), respectively. The total number of cells counted in Fig. 8b was 526. ( c ) Effect of furin inhibitor on localization of furin and LRP1-ICD at 4 h after 0 µM (control) or 30 µM NMDA (NMDA) treatment with or without 10 µM furin inhibitor. The cells were immunostained with anti-furin and anti-β-chain of LRP1/ICD antibodies. Representative images are shown from one experiment. The scale bar represents 30 µm. ( d ) The number of LRP1-ICD immune-positive cells that co-localized with furin in the perinuclear region was counted. Results are expressed as the means ± SD of 4 independent experiments. *Significant difference from the NMDA- and furin-untreated group (NMDA [0] and furin inhibitor [−]) (P < 0.05). # Significant difference from the NMDA-treated and furin inhibitor-untreated group (NMDA [30] and furin inhibitor [−]) (P < 0.05). The range of the number of counted cells in NMDA [0]-furin inhibitor [−], NMDA [0]-furin inhibitor [+], NMDA [30]-furin inhibitor [−], and NMDA [30]-furin inhibitor [+], were 17–27/experiment (the total number of cells counted: 91), 17–33/experiment (total: 110), 27–63/experiment (total: 179), and 19–40/experiment (total: 111), respectively. The total number of cells counted in Fig. 8d was 491.

Article Snippet: For immunocytochemistry, neurons (10 DIV) on glass coverslips pre-coated with poly-D-lysine were washed with PBS (300 μl) twice for 5 min each time and fixed with 4% paraformaldehyde at room temperature for 10 min. After having been washed twice with PBS (300 μl) for 5 min each time, the neurons were made permeable with 0.2% TritonX-100 in PBS (300 μl) by incubation at room temperature for 10 min and then blocked with a mixture of 10% normal goat serum, 1% BSA, and 0.2% Triton X-100 in PBS (300 μl) at room temperature for 1 h. The neurons were incubated with rabbit anti-LRP1 (dilution, 1:2000; 2703-1, Epitomics), goat anti-LRP1 [C II] (dilution, 1:100; AF2368, R&D), mouse anti-TGN46 (dilution 1:200, ab2809, Abcam) or mouse anti-furin (dilution 1:500; sc-133141, Santa cruz) in PBS containing a mixture of 10% normal goat or donkey serum, 1% BSA, and 0.2% Triton X-100 (300 μl) at room temperature for 1 h. The cells were washed 3 times with PBS (300 μl) for 5 min, and then incubated with Alexa Fluor 488-conjugated donkey anti-rabbit IgG (dilution 1:200, Thermo Fisher) and Alexa Fluor 594-conjugated goat anti-mouse IgG (dilution 1:200, Thermo Fisher), and donkey anti-goat IgG at room temperature for 1 h. Subsequently, the neurons were washed 3 times, 5 min each time, with 300 μl of PBS containing Hoechst33342 (0.5 μg/ml, 346-07951, Dojindo, Kumamoto, Japan) and mounted with Fluoromount/Plus (DiagnosticBioSystems, Pleasanton,CA).

Techniques:

Journal: Frontiers in Immunology

Article Title: Extracellular HMGB1 Impairs Macrophage-Mediated Efferocytosis by Suppressing the Rab43-Controlled Cell Surface Transport of CD91

doi: 10.3389/fimmu.2022.767630

Figure Lengend Snippet: rHMGB1 suppressed CD91 expression on the cell surface. The bone marrow-derived macrophages (BMDMs) were treated with 2 μg/ml rHMGB1 for 12 h. (A) The CD91 level on the cell surface was detected using flow cytometry. * P < 0.05 versus the phosphate-buffered saline (PBS)-treated group. (B) Rab43-C or Rab43-cKO BMDMs were incubated with CD91-blocking antibody or IgG for 1 h, and then engulfment of apoptotic cells was measured by flow cytometry. * P < 0.05 versus the Rab43-C/IgG group. (C) Confocal laser-scanning microscopy (CLSM) to determine the subcellular localization of CD91. The BMDMs were stained with the anti-CD91 antibody. Red, CD91; blue, DAPI. Scale bars = 10.0 μm. (D) Co-localization of endogenous CD91 with the ER marker Calnexin. The BMDMs cultured in glass-bottom dishes were stained with anti-CD91 antibody and anti-Calnexin antibodies. Red, CD91; green, Calnexin; blue, DAPI. Scale bars = 10.0 μm. (E) Co-localization of endogenous CD91 with the Golgi body. The BMDMs were fixed with 4% formaldehyde solution for 10 min. A sufficient volume of Dual Detection Reagent was added to cover the monolayer cells (1:100 dilution). After blocking for 1 h, the cells were stained with anti-CD91 antibody. Red, CD91; green, Golgi body; blue, DAPI. Scale bars = 10.0 μm.

Article Snippet: The antibodies used include the PE anti-mouse CD91 Antibody (Bioss, Beijing, China; #bs-10920R-PE), APC anti-mouse F4/80 Antibody (BioLegend, #123116), FITC anti-mouse F4/80 Antibody (BioLegend, #123108), APC/Cyanine7 anti-mouse F4/80 Antibody (BioLegend, #123118), PE/Cyanine7 anti-mouse Ly-6G/Ly-6C (Gr-1) Antibody (BioLegend, #108416), anti-CD16/32 Antibody (BioLegend, #101302), APC anti-mouse CD11c Antibody (BioLegend, #117310), PerCP labeled Anti-Mouse/Human CD11b Antibody (Elabscience, Wuhan, China; #E-AB-F1081F), Anti-RAB43 Antibody (Santa Cruz Biotechnology, #sc-515460), Anti-β-Actin Antibody (CST, #4970S), Anti-CD91 Antibody (Abcam, #ab92544), Calnexin Antibody (Thermo Fisher Scientific, AF18, #MA3-027), Alexa Fluor ® 488-conjugated AffiniPure Goat Anti-Mouse IgG (Jackson Immunoresearch, West Grove, PA, USA; #115-545-003), Alexa Fluor ® 594-conjugated AffiniPure Goat Anti-Rabbit IgG (Jackson Immunoresearch, #111-585-003), Anti-F4/80 Micro-Beads UltraPure, mouse (Miltenyi Biotec, #130-110-443), Anti-FLAG Antibody (CST, #14793), Anti-GFP Antibody (CST, #2956), Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb (HRP Conjugate) (CST, #5127), anti-HMGB1 Neutralizing antibody (Arigo, #ARG66714), ATP1A1 Polyclonal Antibody (Proteintech, #14418-1-AP), BV421 anti-mouse MerTK Antibody (BD Biosciences, #747837), and PE anti-mouse CD36 Antibody (BioLegend, #102605).

Techniques: Expressing, Derivative Assay, Flow Cytometry, Incubation, Blocking Assay, Confocal Laser Scanning Microscopy, Staining, Marker, Cell Culture

Journal: Frontiers in Immunology

Article Title: Extracellular HMGB1 Impairs Macrophage-Mediated Efferocytosis by Suppressing the Rab43-Controlled Cell Surface Transport of CD91

doi: 10.3389/fimmu.2022.767630

Figure Lengend Snippet: Effects of Rab43 knockout on the cell surface transport of endogenous CD91. (A) The CD91 proteins on the cell surface were analyzed using flow cytometry. The bone marrow-derived macrophages (BMDMs) were isolated from Rab43-C and Rab43-cKO mice. * P < 0.05 versus the Rab43-C group. (B) The CD91 levels on the cell surface of BALF samples. The Rab43-C and Rab43-cKO mice were treated with LPS for 1 (upper panel) and 3 days (lower panel). The macrophages (F4/80 + CD11c + CD11b − ) in the BALF samples were also analyzed. * P < 0.05 versus the Rab43-C group. (C) The expression of total, cytoplasmic, and membrane CD91 in Rab43-C and Rab43-cKO BMDMs measured using western blot analyses. *P < 0.05 versus Rab43-C group. ns, not statistically significant.

Article Snippet: The antibodies used include the PE anti-mouse CD91 Antibody (Bioss, Beijing, China; #bs-10920R-PE), APC anti-mouse F4/80 Antibody (BioLegend, #123116), FITC anti-mouse F4/80 Antibody (BioLegend, #123108), APC/Cyanine7 anti-mouse F4/80 Antibody (BioLegend, #123118), PE/Cyanine7 anti-mouse Ly-6G/Ly-6C (Gr-1) Antibody (BioLegend, #108416), anti-CD16/32 Antibody (BioLegend, #101302), APC anti-mouse CD11c Antibody (BioLegend, #117310), PerCP labeled Anti-Mouse/Human CD11b Antibody (Elabscience, Wuhan, China; #E-AB-F1081F), Anti-RAB43 Antibody (Santa Cruz Biotechnology, #sc-515460), Anti-β-Actin Antibody (CST, #4970S), Anti-CD91 Antibody (Abcam, #ab92544), Calnexin Antibody (Thermo Fisher Scientific, AF18, #MA3-027), Alexa Fluor ® 488-conjugated AffiniPure Goat Anti-Mouse IgG (Jackson Immunoresearch, West Grove, PA, USA; #115-545-003), Alexa Fluor ® 594-conjugated AffiniPure Goat Anti-Rabbit IgG (Jackson Immunoresearch, #111-585-003), Anti-F4/80 Micro-Beads UltraPure, mouse (Miltenyi Biotec, #130-110-443), Anti-FLAG Antibody (CST, #14793), Anti-GFP Antibody (CST, #2956), Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb (HRP Conjugate) (CST, #5127), anti-HMGB1 Neutralizing antibody (Arigo, #ARG66714), ATP1A1 Polyclonal Antibody (Proteintech, #14418-1-AP), BV421 anti-mouse MerTK Antibody (BD Biosciences, #747837), and PE anti-mouse CD36 Antibody (BioLegend, #102605).

Techniques: Knock-Out, Flow Cytometry, Derivative Assay, Isolation, Expressing, Western Blot

Journal: Frontiers in Immunology

Article Title: Extracellular HMGB1 Impairs Macrophage-Mediated Efferocytosis by Suppressing the Rab43-Controlled Cell Surface Transport of CD91

doi: 10.3389/fimmu.2022.767630

Figure Lengend Snippet: Rab43 regulates the transport of CD91 to the cell surface. (A) Localization of endogenous CD91 in Rab43-C and Rab43-cKO bone marrow-derived macrophages (BMDMs). The Rab43-C and Rab43-cKO BMDMs were stained with anti-CD91 antibody. Red, CD91; blue, DAPI. Scale bars = 10.0 μm. (B) Co-localization of endogenous CD91 with the ER marker Calnexin. The Rab43-C and Rab43-cKO BMDMs cultured in glass-bottom dishes were stained with anti-CD91 antibody and anti-Calnexin antibodies. Red, CD91; green, Calnexin; blue, DAPI. Scale bars = 10.0 μm. (C) Co-localization of endogenous CD91 with the Golgi body. The Rab43-C and Rab43-cKO BMDMs cultured in glass-bottom dishes were fixed with 4% formaldehyde solution for 10 min. Dual Detection Reagent was used to stain the Golgi body for 1 h. The cultured cells were then incubated with anti-CD91 antibody. Red, CD91; green, Golgi body; blue, DAPI. Scale bars = 10.0 μm.

Article Snippet: The antibodies used include the PE anti-mouse CD91 Antibody (Bioss, Beijing, China; #bs-10920R-PE), APC anti-mouse F4/80 Antibody (BioLegend, #123116), FITC anti-mouse F4/80 Antibody (BioLegend, #123108), APC/Cyanine7 anti-mouse F4/80 Antibody (BioLegend, #123118), PE/Cyanine7 anti-mouse Ly-6G/Ly-6C (Gr-1) Antibody (BioLegend, #108416), anti-CD16/32 Antibody (BioLegend, #101302), APC anti-mouse CD11c Antibody (BioLegend, #117310), PerCP labeled Anti-Mouse/Human CD11b Antibody (Elabscience, Wuhan, China; #E-AB-F1081F), Anti-RAB43 Antibody (Santa Cruz Biotechnology, #sc-515460), Anti-β-Actin Antibody (CST, #4970S), Anti-CD91 Antibody (Abcam, #ab92544), Calnexin Antibody (Thermo Fisher Scientific, AF18, #MA3-027), Alexa Fluor ® 488-conjugated AffiniPure Goat Anti-Mouse IgG (Jackson Immunoresearch, West Grove, PA, USA; #115-545-003), Alexa Fluor ® 594-conjugated AffiniPure Goat Anti-Rabbit IgG (Jackson Immunoresearch, #111-585-003), Anti-F4/80 Micro-Beads UltraPure, mouse (Miltenyi Biotec, #130-110-443), Anti-FLAG Antibody (CST, #14793), Anti-GFP Antibody (CST, #2956), Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb (HRP Conjugate) (CST, #5127), anti-HMGB1 Neutralizing antibody (Arigo, #ARG66714), ATP1A1 Polyclonal Antibody (Proteintech, #14418-1-AP), BV421 anti-mouse MerTK Antibody (BD Biosciences, #747837), and PE anti-mouse CD36 Antibody (BioLegend, #102605).

Techniques: Derivative Assay, Staining, Marker, Cell Culture, Incubation

Journal: Frontiers in Immunology

Article Title: Extracellular HMGB1 Impairs Macrophage-Mediated Efferocytosis by Suppressing the Rab43-Controlled Cell Surface Transport of CD91

doi: 10.3389/fimmu.2022.767630

Figure Lengend Snippet: Rab43 directly interacts with CD91. (A, B) Co-immunoprecipitation of Rab43 with CD91. (Left panel) RAW264.7 cells were transfected with the pcDNA3.1-3xflag-Rab43 plasmid, and the cell lysates were co-immunoprecipitated with anti-FLAG antibody and then immunoblotted with anti-CD91 antibody. (Right panel) RAW264.7 cells were transfected with the pcDNA3.1-GFP-Rab43 plasmid, and the cell lysates were co-immunoprecipitated with anti-CD91 antibody and then immunoblotted with anti-GFP antibody. (C, D) RAW264.7 cells were transfected with plasmids expressing GFP-Rab43 WT and subsequently incubated with anti-CD91 antibody or anti-Calnexin antibodies. Green, GFP-Rab43-C; red, CD91 or Calnexin; blue, DAPI. Scale bars = 10.0 μm.

Article Snippet: The antibodies used include the PE anti-mouse CD91 Antibody (Bioss, Beijing, China; #bs-10920R-PE), APC anti-mouse F4/80 Antibody (BioLegend, #123116), FITC anti-mouse F4/80 Antibody (BioLegend, #123108), APC/Cyanine7 anti-mouse F4/80 Antibody (BioLegend, #123118), PE/Cyanine7 anti-mouse Ly-6G/Ly-6C (Gr-1) Antibody (BioLegend, #108416), anti-CD16/32 Antibody (BioLegend, #101302), APC anti-mouse CD11c Antibody (BioLegend, #117310), PerCP labeled Anti-Mouse/Human CD11b Antibody (Elabscience, Wuhan, China; #E-AB-F1081F), Anti-RAB43 Antibody (Santa Cruz Biotechnology, #sc-515460), Anti-β-Actin Antibody (CST, #4970S), Anti-CD91 Antibody (Abcam, #ab92544), Calnexin Antibody (Thermo Fisher Scientific, AF18, #MA3-027), Alexa Fluor ® 488-conjugated AffiniPure Goat Anti-Mouse IgG (Jackson Immunoresearch, West Grove, PA, USA; #115-545-003), Alexa Fluor ® 594-conjugated AffiniPure Goat Anti-Rabbit IgG (Jackson Immunoresearch, #111-585-003), Anti-F4/80 Micro-Beads UltraPure, mouse (Miltenyi Biotec, #130-110-443), Anti-FLAG Antibody (CST, #14793), Anti-GFP Antibody (CST, #2956), Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb (HRP Conjugate) (CST, #5127), anti-HMGB1 Neutralizing antibody (Arigo, #ARG66714), ATP1A1 Polyclonal Antibody (Proteintech, #14418-1-AP), BV421 anti-mouse MerTK Antibody (BD Biosciences, #747837), and PE anti-mouse CD36 Antibody (BioLegend, #102605).

Techniques: Immunoprecipitation, Transfection, Plasmid Preparation, Expressing, Incubation