Journal: Acta biochimica et biophysica Sinica

Article Title: RanBPM is an acetylcholinesterase-interacting protein that translocates into the nucleus during apoptosis.

doi: 10.1093/abbs/gmp082

Figure Lengend Snippet: Figure 1 AChE interacts with RanBPM (A) The self-expression assay of the bait. AH109/pGBKT7-AChEC-terminal cells were cultured on SD/-Trp plate or SD/-Trp/-His plates. (B) Library screening. AH109 clones transfected with pGBKT7-AChEC-terminal and human fetal brain library were cultured on SD/-Ade/-His/-Leu/-Trp/X-a-gal plates. The arrows indicate the positive clones (blue clones). (C) The scheme of the two positive clones (A and C6). The putative SPRY domain (amino acids 212–333) is indicated. (D) Clones A and C6 showed strong lacZ reporter activation. Pos., a clone consisting of BD-p53 and AD-SV40 T-antigen was used as the positive control. Neg., a combination of BD-laminC and AD-SV40 T-antigen was used as the negative control; b-galactosidase activity is expressed as a percentage of the positive control. The data are represented as the mean+ SD of three independent transformations. (E,F) HEK293T cells transfected with the indicated expression plasmid for 24 h; the whole-cell lysates were immunoprecipitated with anti-Myc antibody and analyzed by immunoblotting with anti-HA antibody (top panel). Separate aliquots of the lysates were immunoblotted with anti-HA (middle panel) or anti-Myc (bottom panel) to confirm the expression of HA-RanBPM or Myc-AChEC-terminal (or Myc-AChE), respectively. (G) Kidney tissues from the rat model of ischemia/reperfusion injury were immunoprecipitated using the anti-AChE antibody (E70), and analyzed by immunoblotting with the anti-RanBPM antibody (top panel). Separate aliquots of the lysates were immunoblotted with anti-RanBPM (middle panel) or anti-AChE (E70) (bottom panel) to confirm the expression of RanBPM or AChE, respectively, in the rat kidney tissues.

Article Snippet: The primary antibodies used were as follows: anti-RanBPM antibody (NB100-1281; Novus Biologicals) (1:1000), anti-AChE antibody (the antibody against AChE was prepared in the rabbit [37]) (1:1000), anti-Myc antibody (1:5000), anti-AChE antibody (1:1000), anti-GFP antibody (sc-9996; Santa Cruz) (1:1000), anti-HA antibody (sc-805; Santa Cruz) (1:500), anti-caspase 3 antibody (Cell Signalling, #9662) (1:500), anti-histone 3 antibody (ab1791; ABCAM) (1:3000), anti-tubulin antibody (T6074; Sigma) (1:5000), anti-actin antibody (A5441; Sigma) (1:5000).

Techniques: Expressing, Cell Culture, Library Screening, Clone Assay, Transfection, Activation Assay, Positive Control, Negative Control, Activity Assay, Plasmid Preparation, Immunoprecipitation, Western Blot

Journal: Acta biochimica et biophysica Sinica

Article Title: RanBPM is an acetylcholinesterase-interacting protein that translocates into the nucleus during apoptosis.

doi: 10.1093/abbs/gmp082

Figure Lengend Snippet: Figure 3 Subcellular distribution of RanBPM and AChE (A,B) Fractionation of HEK293T cell extracts. HEK293T cells were transfected with the indicated expression plasmids. At 24 h after transfection, the cells were fractionated into nuclear (N) and cytoplasmic (C) fractions and analyzed directly by immunoblotting with the anti-HA (first panel) or anti-Myc antibody (second panel). The purity of the nuclear and cytoplasmic fractions was examined by immunoblotting with anti-histone 3 (fourth panel) and anti-a-tubulin antibody (third panel), respectively. (C) Determination of co-localization of AChE and RanBPM by immunofluorescence. HEK293T cells were transfected with Myc-RanBPM and GFP-AChE. At 24 h after transfection, the cells were fixed and incubated with anti-Myc (red), followed by incubation with Rhodamine-conjugated secondary antibodies. The cells were examined by fluorescence microscopy.

Article Snippet: The primary antibodies used were as follows: anti-RanBPM antibody (NB100-1281; Novus Biologicals) (1:1000), anti-AChE antibody (the antibody against AChE was prepared in the rabbit [37]) (1:1000), anti-Myc antibody (1:5000), anti-AChE antibody (1:1000), anti-GFP antibody (sc-9996; Santa Cruz) (1:1000), anti-HA antibody (sc-805; Santa Cruz) (1:500), anti-caspase 3 antibody (Cell Signalling, #9662) (1:500), anti-histone 3 antibody (ab1791; ABCAM) (1:3000), anti-tubulin antibody (T6074; Sigma) (1:5000), anti-actin antibody (A5441; Sigma) (1:5000).

Techniques: Fractionation, Transfection, Expressing, Western Blot, Incubation, Fluorescence, Microscopy

Journal: Acta biochimica et biophysica Sinica

Article Title: RanBPM is an acetylcholinesterase-interacting protein that translocates into the nucleus during apoptosis.

doi: 10.1093/abbs/gmp082

Figure Lengend Snippet: Figure 4 Subcellular distribution of RanBPM and AChE after cisplatin treatment (A) HEK293T cell viability was assessed by MTT after treatment with different concentrations of cisplatin for 24 h. The graph shows the mean+ SE of three independent experiments. (B) The transcription level of p73a gene detected by RT–PCR. HEK293T cells were treated with different concentrations of cisplatin for 24 h and then total mRNA was extracted for RT–PCR. (C) Cisplatin of 100 mM or higher induced DNA fragmentation. (D) The amount of RanBPM and AChE in the nuclear fractions increased after treatment with cisplatin. After cotransfection with pCMV-Myc-RanBPM and pEGFP-C1-AChE for 24 h, HEK293T cells were treated with the indicated concentrations of cisplatin for another 24 h. Then, the cells were fractionated into nuclear (N) and cytoplasmic (C) fractions. The cell fractions and the whole-cell lysates were immunoblotted with anti-Myc (left line) or anti-GFP (light line). The graph shows the densitometric values of the proteins in the cytoplasm versus those in the nuclei.

Article Snippet: The primary antibodies used were as follows: anti-RanBPM antibody (NB100-1281; Novus Biologicals) (1:1000), anti-AChE antibody (the antibody against AChE was prepared in the rabbit [37]) (1:1000), anti-Myc antibody (1:5000), anti-AChE antibody (1:1000), anti-GFP antibody (sc-9996; Santa Cruz) (1:1000), anti-HA antibody (sc-805; Santa Cruz) (1:500), anti-caspase 3 antibody (Cell Signalling, #9662) (1:500), anti-histone 3 antibody (ab1791; ABCAM) (1:3000), anti-tubulin antibody (T6074; Sigma) (1:5000), anti-actin antibody (A5441; Sigma) (1:5000).

Techniques: Reverse Transcription Polymerase Chain Reaction, Cotransfection

Journal: Acta biochimica et biophysica Sinica

Article Title: RanBPM is an acetylcholinesterase-interacting protein that translocates into the nucleus during apoptosis.

doi: 10.1093/abbs/gmp082

Figure Lengend Snippet: Figure 5 Nuclear distribution of endogenous RanBPM and AChE after long-term culture (A) Cleaved caspase 3 was detected in long-term cultured HEK293T cells. HEK293T cells were cultured without medium changed for 3 days (3D) or 7 days (7D), and then the whole-cell lysates were immunoblotted with anti-capspase 3, or anti-b-actin antibody. (B) The amount of endogenous RanBPM and AChE in the nuclear fractions was increased after long-term culture. HEK293T cells were cultured without medium changed for 2 days (2D), 5 days (5D) or 8 days (8D), and then the nuclear fractions were immunoblotted with anti-AChE (first panel) or anti-RanBPM antibody (second panel, NB 100–1281). The purity of the nuclear and cytoplasmic fractions was examined by immunoblotting with anti-a-tubulin (third panel) or anti-histone 3 antibody (fourth panel), respectively.

Article Snippet: The primary antibodies used were as follows: anti-RanBPM antibody (NB100-1281; Novus Biologicals) (1:1000), anti-AChE antibody (the antibody against AChE was prepared in the rabbit [37]) (1:1000), anti-Myc antibody (1:5000), anti-AChE antibody (1:1000), anti-GFP antibody (sc-9996; Santa Cruz) (1:1000), anti-HA antibody (sc-805; Santa Cruz) (1:500), anti-caspase 3 antibody (Cell Signalling, #9662) (1:500), anti-histone 3 antibody (ab1791; ABCAM) (1:3000), anti-tubulin antibody (T6074; Sigma) (1:5000), anti-actin antibody (A5441; Sigma) (1:5000).

Techniques: Cell Culture, Western Blot

Journal: Frontiers in Immunology

Article Title: Histamine Regulates the Inflammatory Profile of SOD1-G93A Microglia and the Histaminergic System Is Dysregulated in Amyotrophic Lateral Sclerosis

doi: 10.3389/fimmu.2017.01689

Figure Lengend Snippet: SOD1-G93A microglia express histaminergic receptors. (A) Primary microglia from SOD1-G93A mice were stained with anti-CD11b (red) and anti-H1R, anti-H2R, anti-H3R, and anti-H4R (green). Höechst 33258 was used for nuclei (scale bar 50 µm). In inserts, wild-type (WT) microglia (scale bar 50 µm). (B) Equal amounts of WT and SOD1-G93A primary microglia total lysates were subjected to western blotting with anti-H1R, anti-H2R, anti-H3R, and anti-H4R. Anti-GAPDH was used for protein normalization. Data represent mean ± SEM of n = 3 independent experiments. Statistical significance was calculated by Student’s t -test, as referred to WT cells, * p < 0.05.

Article Snippet: Antibodies for WB and IF were rabbit anti-HDC (1:500 WB; 1:200 IF, Abcam, USA); rabbit anti-HNMT (1:200 WB, IF, Atlas, Sweden); rabbit anti-DAO (1:200 WB and IF, Bioss, USA); rabbit anti-H1R (1:500 WB, 1:200 IF, Alomone, Israel); rabbit anti-H2R (1:500 WB and 1:200 IF, Alomone); rabbit anti-H3R (1:500 WB, 1:200 IF, Alomone); rabbit anti-H4R (1:500 WB, 1:200 IF, Santa Cruz); mouse anti-NOX2/gp91 phox (1:1,000 WB, BD Transduction Laboratories, USA); rabbit anti-phospho-NF-κB p65 (Ser536) (1:500 WB, CST, USA); rabbit anti-NF-κB p65 (1:500 WB, CST); rabbit anti-phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (1:1,000 WB, CST); mouse anti-p44/42 MAPK (ERK1/2) (L34F12) (1:1,000 WB, CST); rabbit anti-phospho-p38 MAPK (Thr180/Tyr182) (1:500 WB, CST); mouse anti-p38 MAPK (A-12) (1:500 WB, Santa Cruz); rabbit anti-Arginase 1 (ARG1) (1:700 WB, Abcam); rabbit anti-P2Y12 (1:200 WB, Anaspec, USA); rat anti-CD11b (1:200 IF, AbD Serotec, USA); rabbit anti-Iba1 (1:500, WB, IF, Wako, USA); rat anti-CD68 (1:500, WB, IF, AbD Serotec); rabbit anti-CD163 (1:100 WB, Santa Cruz); rabbit anti-CD206 (1:500 WB, Abcam); mouse anti-GAPDH (1:2,500 WB, Calbiochem, USA).

Techniques: Staining, Western Blot

Journal: Frontiers in Immunology

Article Title: Histamine Regulates the Inflammatory Profile of SOD1-G93A Microglia and the Histaminergic System Is Dysregulated in Amyotrophic Lateral Sclerosis

doi: 10.3389/fimmu.2017.01689

Figure Lengend Snippet: MAPKs are activated by histamine in SOD1-G93A microglia. Wild-type (WT) and SOD1-G93A primary microglia were treated with histamine (100 µM) for 15 min–1 h–6 h and equal amounts of total lysates were subjected to SDS-PAGE, western blotting, and immunoreactions with anti-p-p38 and anti-p-38 (A) or with anti-pERK and anti-ERK (B) . (C) SOD1-G93A microglia stimulated with histamine (100 µM) for 1 h in the presence of specific antagonists for H1R (orphenadrine, 10 µM), H2R (ranitidine, 10 µM), H3R (thioperamide, 5 µM), or H4R (JNJ-7777120, 5 µM) were subjected to immunoreactions with anti-pERK and anti-ERK. Anti-GAPDH was used for protein normalization. HA, histamine. Data represent mean ± SEM of n = 4 independent experiments. Statistical significance was calculated by ANOVA followed by Post Hoc Tukey’s test, as referred to ctrl cells, * p < 0.05 or to histamine-treated cells, # p < 0.05.

Article Snippet: Antibodies for WB and IF were rabbit anti-HDC (1:500 WB; 1:200 IF, Abcam, USA); rabbit anti-HNMT (1:200 WB, IF, Atlas, Sweden); rabbit anti-DAO (1:200 WB and IF, Bioss, USA); rabbit anti-H1R (1:500 WB, 1:200 IF, Alomone, Israel); rabbit anti-H2R (1:500 WB and 1:200 IF, Alomone); rabbit anti-H3R (1:500 WB, 1:200 IF, Alomone); rabbit anti-H4R (1:500 WB, 1:200 IF, Santa Cruz); mouse anti-NOX2/gp91 phox (1:1,000 WB, BD Transduction Laboratories, USA); rabbit anti-phospho-NF-κB p65 (Ser536) (1:500 WB, CST, USA); rabbit anti-NF-κB p65 (1:500 WB, CST); rabbit anti-phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (1:1,000 WB, CST); mouse anti-p44/42 MAPK (ERK1/2) (L34F12) (1:1,000 WB, CST); rabbit anti-phospho-p38 MAPK (Thr180/Tyr182) (1:500 WB, CST); mouse anti-p38 MAPK (A-12) (1:500 WB, Santa Cruz); rabbit anti-Arginase 1 (ARG1) (1:700 WB, Abcam); rabbit anti-P2Y12 (1:200 WB, Anaspec, USA); rat anti-CD11b (1:200 IF, AbD Serotec, USA); rabbit anti-Iba1 (1:500, WB, IF, Wako, USA); rat anti-CD68 (1:500, WB, IF, AbD Serotec); rabbit anti-CD163 (1:100 WB, Santa Cruz); rabbit anti-CD206 (1:500 WB, Abcam); mouse anti-GAPDH (1:2,500 WB, Calbiochem, USA).

Techniques: SDS Page, Western Blot

Journal: Frontiers in Immunology

Article Title: Histamine Regulates the Inflammatory Profile of SOD1-G93A Microglia and the Histaminergic System Is Dysregulated in Amyotrophic Lateral Sclerosis

doi: 10.3389/fimmu.2017.01689

Figure Lengend Snippet: NF-κB pathway is modulated by histamine in SOD1-G93A microglia. (A) Total RNA was extracted from SOD1-G93A microglia stimulated with histamine (100 µM) for 6–18 h and the expression profiles of IL-10, IL-6, and IL-1β were examined by qRT-PCR. (B) Wild-type (WT) and SOD1-G93A microglia exposed to 100 µM histamine for 1–6–18 h were subjected to immunoreactions with anti-pNF-κB and anti-NF-κB. (C) SOD1-G93A microglia stimulated with histamine (100 µM) for 6 h in the presence of H1R–H2R–H3R–H4R antagonists were subjected to immunoreactions with anti-pNF-κB and anti-NF-κB. (D) SOD1-G93A microglia treated with histamine (100 µM) for 6 h in the presence of specific inhibitors of pERK (PD98059, 100 µM) and pp38 (SB203580, 20 µM) were subjected to immunoreactions with anti-pNF-κB and anti-NF-κB. Anti-GAPDH was used for protein normalization. HA, histamine. Data represent mean ± SEM of n = 4 independent experiments. Statistical significance was calculated by ANOVA followed by Post Hoc Tukey’s test, as referred to ctrl cells, * p < 0.05 or to histamine-treated cells, # p < 0.05.

Article Snippet: Antibodies for WB and IF were rabbit anti-HDC (1:500 WB; 1:200 IF, Abcam, USA); rabbit anti-HNMT (1:200 WB, IF, Atlas, Sweden); rabbit anti-DAO (1:200 WB and IF, Bioss, USA); rabbit anti-H1R (1:500 WB, 1:200 IF, Alomone, Israel); rabbit anti-H2R (1:500 WB and 1:200 IF, Alomone); rabbit anti-H3R (1:500 WB, 1:200 IF, Alomone); rabbit anti-H4R (1:500 WB, 1:200 IF, Santa Cruz); mouse anti-NOX2/gp91 phox (1:1,000 WB, BD Transduction Laboratories, USA); rabbit anti-phospho-NF-κB p65 (Ser536) (1:500 WB, CST, USA); rabbit anti-NF-κB p65 (1:500 WB, CST); rabbit anti-phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (1:1,000 WB, CST); mouse anti-p44/42 MAPK (ERK1/2) (L34F12) (1:1,000 WB, CST); rabbit anti-phospho-p38 MAPK (Thr180/Tyr182) (1:500 WB, CST); mouse anti-p38 MAPK (A-12) (1:500 WB, Santa Cruz); rabbit anti-Arginase 1 (ARG1) (1:700 WB, Abcam); rabbit anti-P2Y12 (1:200 WB, Anaspec, USA); rat anti-CD11b (1:200 IF, AbD Serotec, USA); rabbit anti-Iba1 (1:500, WB, IF, Wako, USA); rat anti-CD68 (1:500, WB, IF, AbD Serotec); rabbit anti-CD163 (1:100 WB, Santa Cruz); rabbit anti-CD206 (1:500 WB, Abcam); mouse anti-GAPDH (1:2,500 WB, Calbiochem, USA).

Techniques: Expressing, Quantitative RT-PCR

Journal: Frontiers in Immunology

Article Title: Histamine Regulates the Inflammatory Profile of SOD1-G93A Microglia and the Histaminergic System Is Dysregulated in Amyotrophic Lateral Sclerosis

doi: 10.3389/fimmu.2017.01689

Figure Lengend Snippet: Histamine modulates inflammation in SOD1-G93A microglia. Wild-type (WT) and SOD1-G93A primary microglia were exposed to 100 µM histamine for 1–6–18 h and equal amounts of total lysates subjected to western blotting (WB) and immunoreactions with anti-NADPH oxidase 2 (NOX2) (A) , anti-ARG-1 (B) . SOD1-G93A microglia stimulated with histamine (100 µM) in the presence of H1R–H2R–H3R–H4R antagonists for 6 h were subjected to immunoreactions with anti-NOX2 (C) or anti-ARG-1 (D) . SOD1-G93A primary microglia were exposed to 100 µM histamine for 1–6–18 h and equal amounts of total lysates subjected to WB and immunoreactions with anti-CD163 (E) or anti-CD206 (F) . Anti-GAPDH was used for protein normalization. HA, histamine. Data represent mean ± SEM of n = 4 independent experiments. Statistical significance was calculated by ANOVA followed by Post Hoc Tukey’s test, as referred to ctrl cells, * p < 0.05 or to histamine-treated cells, # p < 0.05.

Article Snippet: Antibodies for WB and IF were rabbit anti-HDC (1:500 WB; 1:200 IF, Abcam, USA); rabbit anti-HNMT (1:200 WB, IF, Atlas, Sweden); rabbit anti-DAO (1:200 WB and IF, Bioss, USA); rabbit anti-H1R (1:500 WB, 1:200 IF, Alomone, Israel); rabbit anti-H2R (1:500 WB and 1:200 IF, Alomone); rabbit anti-H3R (1:500 WB, 1:200 IF, Alomone); rabbit anti-H4R (1:500 WB, 1:200 IF, Santa Cruz); mouse anti-NOX2/gp91 phox (1:1,000 WB, BD Transduction Laboratories, USA); rabbit anti-phospho-NF-κB p65 (Ser536) (1:500 WB, CST, USA); rabbit anti-NF-κB p65 (1:500 WB, CST); rabbit anti-phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (1:1,000 WB, CST); mouse anti-p44/42 MAPK (ERK1/2) (L34F12) (1:1,000 WB, CST); rabbit anti-phospho-p38 MAPK (Thr180/Tyr182) (1:500 WB, CST); mouse anti-p38 MAPK (A-12) (1:500 WB, Santa Cruz); rabbit anti-Arginase 1 (ARG1) (1:700 WB, Abcam); rabbit anti-P2Y12 (1:200 WB, Anaspec, USA); rat anti-CD11b (1:200 IF, AbD Serotec, USA); rabbit anti-Iba1 (1:500, WB, IF, Wako, USA); rat anti-CD68 (1:500, WB, IF, AbD Serotec); rabbit anti-CD163 (1:100 WB, Santa Cruz); rabbit anti-CD206 (1:500 WB, Abcam); mouse anti-GAPDH (1:2,500 WB, Calbiochem, USA).

Techniques: Western Blot

Journal: Frontiers in Immunology

Article Title: Histamine Regulates the Inflammatory Profile of SOD1-G93A Microglia and the Histaminergic System Is Dysregulated in Amyotrophic Lateral Sclerosis

doi: 10.3389/fimmu.2017.01689

Figure Lengend Snippet: Histamine induces migration in SOD1-G93A microglia. (A) Wild-type (WT) and SOD1-G93A primary microglia were exposed to 100 µM histamine for 1–6–18 h and equal amounts of total lysates subjected to western blotting and immunoreactions with anti-P2Y12. Anti-GAPDH was used for protein normalization. (B) SOD1-G93A microglia were stimulated with 100 µM histamine in the presence or absence of H1R–H2R–H3R–H4R antagonists or P2Y12 antagonist (MRS2395, 10 µM) for 18 h and stained with anti-CD11b (green). Scale bar 50 µm. The number of migrating cells was then quantified. (C) WT microglia were stimulated with 100 microM histamine in the presence or absence of H1R-H2R-H3R-H4R antagonists for 18 h and the number of migrating cells was quantified, HA, histamine. Data represent mean ± SEM of n = 3 independent experiments. Statistical significance was calculated by ANOVA followed by Post Hoc Tukey’s test, as referred to ctrl cells, * p < 0.05 or to histamine-treated cells, # p < 0.05.

Article Snippet: Antibodies for WB and IF were rabbit anti-HDC (1:500 WB; 1:200 IF, Abcam, USA); rabbit anti-HNMT (1:200 WB, IF, Atlas, Sweden); rabbit anti-DAO (1:200 WB and IF, Bioss, USA); rabbit anti-H1R (1:500 WB, 1:200 IF, Alomone, Israel); rabbit anti-H2R (1:500 WB and 1:200 IF, Alomone); rabbit anti-H3R (1:500 WB, 1:200 IF, Alomone); rabbit anti-H4R (1:500 WB, 1:200 IF, Santa Cruz); mouse anti-NOX2/gp91 phox (1:1,000 WB, BD Transduction Laboratories, USA); rabbit anti-phospho-NF-κB p65 (Ser536) (1:500 WB, CST, USA); rabbit anti-NF-κB p65 (1:500 WB, CST); rabbit anti-phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (1:1,000 WB, CST); mouse anti-p44/42 MAPK (ERK1/2) (L34F12) (1:1,000 WB, CST); rabbit anti-phospho-p38 MAPK (Thr180/Tyr182) (1:500 WB, CST); mouse anti-p38 MAPK (A-12) (1:500 WB, Santa Cruz); rabbit anti-Arginase 1 (ARG1) (1:700 WB, Abcam); rabbit anti-P2Y12 (1:200 WB, Anaspec, USA); rat anti-CD11b (1:200 IF, AbD Serotec, USA); rabbit anti-Iba1 (1:500, WB, IF, Wako, USA); rat anti-CD68 (1:500, WB, IF, AbD Serotec); rabbit anti-CD163 (1:100 WB, Santa Cruz); rabbit anti-CD206 (1:500 WB, Abcam); mouse anti-GAPDH (1:2,500 WB, Calbiochem, USA).

Techniques: Migration, Western Blot, Staining

Journal: Frontiers in Immunology

Article Title: Histamine Regulates the Inflammatory Profile of SOD1-G93A Microglia and the Histaminergic System Is Dysregulated in Amyotrophic Lateral Sclerosis

doi: 10.3389/fimmu.2017.01689

Figure Lengend Snippet: Protein expression levels of H1-H4R in cortex and lumbar spinal cord from wild-type (WT) and SOD1-G93A mice. Cortex (A,C,E,G) and lumbar spinal cord (B,D,F,H) from WT (~20 weeks) and SOD1-G93A mice at different stages of disease (100, 140 days, and end stage) ( n = 4/group) were subjected to SDS-PAGE, western blotting, and immunoreactions with anti-H1R, anti-H2R, anti-H3R, and anti-H4R. Anti-GAPDH was used for protein normalization. Data represent mean ± SEM. Statistical significance was calculated by ANOVA followed by Post Hoc Tukey’s test, as referred to WT mice, * p < 0.05.

Article Snippet: Antibodies for WB and IF were rabbit anti-HDC (1:500 WB; 1:200 IF, Abcam, USA); rabbit anti-HNMT (1:200 WB, IF, Atlas, Sweden); rabbit anti-DAO (1:200 WB and IF, Bioss, USA); rabbit anti-H1R (1:500 WB, 1:200 IF, Alomone, Israel); rabbit anti-H2R (1:500 WB and 1:200 IF, Alomone); rabbit anti-H3R (1:500 WB, 1:200 IF, Alomone); rabbit anti-H4R (1:500 WB, 1:200 IF, Santa Cruz); mouse anti-NOX2/gp91 phox (1:1,000 WB, BD Transduction Laboratories, USA); rabbit anti-phospho-NF-κB p65 (Ser536) (1:500 WB, CST, USA); rabbit anti-NF-κB p65 (1:500 WB, CST); rabbit anti-phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (1:1,000 WB, CST); mouse anti-p44/42 MAPK (ERK1/2) (L34F12) (1:1,000 WB, CST); rabbit anti-phospho-p38 MAPK (Thr180/Tyr182) (1:500 WB, CST); mouse anti-p38 MAPK (A-12) (1:500 WB, Santa Cruz); rabbit anti-Arginase 1 (ARG1) (1:700 WB, Abcam); rabbit anti-P2Y12 (1:200 WB, Anaspec, USA); rat anti-CD11b (1:200 IF, AbD Serotec, USA); rabbit anti-Iba1 (1:500, WB, IF, Wako, USA); rat anti-CD68 (1:500, WB, IF, AbD Serotec); rabbit anti-CD163 (1:100 WB, Santa Cruz); rabbit anti-CD206 (1:500 WB, Abcam); mouse anti-GAPDH (1:2,500 WB, Calbiochem, USA).

Techniques: Expressing, SDS Page, Western Blot