Journal: bioRxiv

Article Title: Rab5 and Alsin regulate stress-activated cytoprotective signaling on mitochondria

doi: 10.1101/200428

Figure Lengend Snippet: BAC GFP-Rab5 cells seeded on a gridded dish were labeled with MitoTracker-Red CMSRox and photoirradiated as before. Cells were fixed after 30 min post-laser treatment and immunostained with specific ZFYVE20 and EEA1 ( A ), and APPL1 and APPL2 antibodies ( B ). ( C ) Quantification of colocalization from untreated and laser-treated cells in ( A ) and ( B ), n = 3. ( D ) Quantification of colocalization between Rab5 and EEA1 as shown in ( A ), n = 3. ( E - F ) BAC GFP-Rab5 cells were treated and processed in the same manner as in ( A ) and immunostained with specific Rabex-5 and Alsin antibodies, respectively. ( G ) Quantification of colocalization from untreated and laser-treated cells in ( E ) and ( F ), n = 3. Scale bars, 10 μm.

Article Snippet: Cells were immunostained with corresponding primary: anti-rabbit Rabenosyn-5/ZFYVE20 (Sigma Aldrich: HPA044878), anti-mouse EEA1 (BD Biosciences: 610457), anti-rabbit TOM20 (Santa Cruz Biotechnology: sc-11415), anti-rabbit APPL1 (Abcam: ab59592), anti-mouse Rab5 (BD Biosciences: 610724), anti-mouse cytochrome c (Abcam: ab6311), and anti-rabbit Alsin (Novus Biological: NBP2-14284) antibodies.

Techniques: Labeling

Journal: bioRxiv

Article Title: Rab5 and Alsin regulate stress-activated cytoprotective signaling on mitochondria

doi: 10.1101/200428

Figure Lengend Snippet: ( A ) BAC GFP-Rab5 cells were seeded on glass coverslip, fixed, and immunostained with specific antibodies against Rabex-5. ( B ) A schematic representation of domain organization of full-length human Alsin. The regulator of chromosome condensation 1 (RCC1), B-cell lymphoma (Dbl) homology (DH) and pleckstrin homology (PH), membrane occupation and recognition nexus (MORN) motif, and vacuolar protein sorting 9 (VPS9) domains are labeled. Three putative GEF domains are annotated. ( C ) Immunostaining of HeLa cells with specific antibodies against Rab5 and Alsin.

Article Snippet: Cells were immunostained with corresponding primary: anti-rabbit Rabenosyn-5/ZFYVE20 (Sigma Aldrich: HPA044878), anti-mouse EEA1 (BD Biosciences: 610457), anti-rabbit TOM20 (Santa Cruz Biotechnology: sc-11415), anti-rabbit APPL1 (Abcam: ab59592), anti-mouse Rab5 (BD Biosciences: 610724), anti-mouse cytochrome c (Abcam: ab6311), and anti-rabbit Alsin (Novus Biological: NBP2-14284) antibodies.

Techniques: Membrane, Labeling, Immunostaining

Journal: bioRxiv

Article Title: Rab5 and Alsin regulate stress-activated cytoprotective signaling on mitochondria

doi: 10.1101/200428

Figure Lengend Snippet: ( A ) Electrophoresis result of the PCR reaction using primers flanking exon 3 and within exon 3. Homozygous deletion was confirmed by the absence of a 2.8 kb in Alsin -/- . ( B ) Protein lysates from WT KOLF and Alsin -/- iPSC were loaded onto SDS-PAGE and probed with antibody against Alsin. A band detected at ∼184 kDa, but absent in Alsin -/- , corresponds to full-length Alsin. ( C ) WT KOLF and Alsin -/- iPSC and neuroprogenitor cells (NPC) were immunostained with pluripotency markers Oct4 and Lin28, and neuroprogenitor markers Sox2 and Pax6, respectively. ( D-E ) WT KOLF and Alsin -/- iPSC-sMN were immunostained with motor neuron markers such as ChAT, HB9, and ISL1, nuclear dye DAPI, and cytoskeletal marker MAP2. ( F ) WT KOLF and Alsin -/- iPSC-sMN were immunostained with specific antibodies against Rab5 and Alsin, along with DAPI (nuclear) and phalloidin (actin) stains. Scale bars, 10 μm.

Article Snippet: Cells were immunostained with corresponding primary: anti-rabbit Rabenosyn-5/ZFYVE20 (Sigma Aldrich: HPA044878), anti-mouse EEA1 (BD Biosciences: 610457), anti-rabbit TOM20 (Santa Cruz Biotechnology: sc-11415), anti-rabbit APPL1 (Abcam: ab59592), anti-mouse Rab5 (BD Biosciences: 610724), anti-mouse cytochrome c (Abcam: ab6311), and anti-rabbit Alsin (Novus Biological: NBP2-14284) antibodies.

Techniques: Electrophoresis, SDS Page, Marker

Journal: bioRxiv

Article Title: Rab5 and Alsin regulate stress-activated cytoprotective signaling on mitochondria

doi: 10.1101/200428

Figure Lengend Snippet: ( A ) Flow chart depicting the different stages and time (in days) from iPSC, to neuroprogenitor cells (NPC), and to generating mature spinal motor neurons (sMN). Figure legends indicate the various small molecules and compounds that are used at different stages. ( B ) WT and Alsin -/- cells were challenged with 100 μM H 2 O 2 for 1 hour. Cells were fixed and immunostained with Rab5 and TOM20 antibodies. Inset images show the representative of the signals from TOM20 and Rab5 under control (left panel) and H 2 O 2 -treated condition (right panel). Scale bars, 10 μm. ( C ) Subcellular fractionation of cytosolic (Cyto) and mitochondrial (Mito) fractions from WT and Alsin -/- iPSC-sMN treated with either PBS (control) or 200 μM H 2 O 2 for 1 hours at 37°C. Protein samples were loaded onto SDS-PAGE and imunoblotted with Rab5, tubulin (cytoplasmic loading control), and TOM20 antibodies by Western blot. ( D ) Cytosolic fractions were prepared as in from WT and Alsin -/- iPSC-sMN challenged with 200 μM H 2 O 2 for 1 hours at 37μC. Densitometric quantification of cytosolic cytochrome c from Western blot probed cytochrome c antibody. Data were collected from three independent experiments. Y-axis corresponds to normalized ratio intensity of experimental to loading control (* p <0.05; ** p <0.005).

Article Snippet: Cells were immunostained with corresponding primary: anti-rabbit Rabenosyn-5/ZFYVE20 (Sigma Aldrich: HPA044878), anti-mouse EEA1 (BD Biosciences: 610457), anti-rabbit TOM20 (Santa Cruz Biotechnology: sc-11415), anti-rabbit APPL1 (Abcam: ab59592), anti-mouse Rab5 (BD Biosciences: 610724), anti-mouse cytochrome c (Abcam: ab6311), and anti-rabbit Alsin (Novus Biological: NBP2-14284) antibodies.

Techniques: Control, Fractionation, SDS Page, Western Blot

Journal: bioRxiv

Article Title: Rab5 and Alsin regulate stress-activated cytoprotective signaling on mitochondria

doi: 10.1101/200428

Figure Lengend Snippet: In normal condition, mitochondria (Mito, red) are elongated and tubular (top left). Rab5 (green) are localized on early endosomes (EE) to assemble Rab5 machinery for endosomal maturation and membrane trafficking. At steady state, we observed some EE transiently making contacts with mitochondria. During oxidative stress (eg. laser- or chemically-induced) that leads to MOMP, mitochondria undergo a dramatic morphological transformation into rounded and swollen structures (top right). This is accompanied by an increase in Rab5-positive endosomes forming MCS with mitochondria of less than <5 nm. Release of apoptotic signal such as cytochrome c from mitochondria into the cytosol is associated with Rab5 translocation to the OMM, which is involved in blocking cytochrome c release and caspase activation. The recruitment and activation of Rab5 on mitochondria depend on the Rab5 GEF Alsin (blue), resulting in a selective recruitment of Rabenosyn-5 (light green), in order to regulate apoptosis and confer cytoprotection.

Article Snippet: Cells were immunostained with corresponding primary: anti-rabbit Rabenosyn-5/ZFYVE20 (Sigma Aldrich: HPA044878), anti-mouse EEA1 (BD Biosciences: 610457), anti-rabbit TOM20 (Santa Cruz Biotechnology: sc-11415), anti-rabbit APPL1 (Abcam: ab59592), anti-mouse Rab5 (BD Biosciences: 610724), anti-mouse cytochrome c (Abcam: ab6311), and anti-rabbit Alsin (Novus Biological: NBP2-14284) antibodies.

Techniques: Membrane, Transformation Assay, Translocation Assay, Blocking Assay, Activation Assay

Journal: PLoS ONE

Article Title: Loss of ALS2/Alsin Exacerbates Motor Dysfunction in a SOD1 H46R -Expressing Mouse ALS Model by Disturbing Endolysosomal Trafficking

doi: 10.1371/journal.pone.0009805

Figure Lengend Snippet: Western blot analysis of the levels of proteins, including ALS2, SOD1, ubiquitin (Ub), polyubiquitin binding protein p62/SQSTM1 (p62), microtubule-associated protein 1-light chain 3 (LC3), peripherin, neurofilament heavy chain (NFH), TAR DNA-binding protein 43-kD (TDP-43), heat-shock protein Hsp70, 20S proteasome subunits (α5, C2, and LMP2), vimentin, and glial fibrillary acidic protein (GFAP), in the lumbo-sacral cord from 8, 12, 16, and 20 week-old mice with four distinct genotypes; wild-type ( Als2 +/+ ), Als2 +/+ ; SOD1 H46R , Als2 +/− ; SOD1 H46R , and Als2 −/− ; SOD1 H46R . Two fractions; 1% Triton X-soluble fraction (TX-soluble; left panels) and 1% Triton X-insoluble/5% SDS-soluble fraction (TX-insoluble; right panels) were analyzed. SOD1_mono and SOD1_HMW represent monomeric and high molecular-weight (aggregated) forms of SOD1, respectively. Ub_mono and Ub_HMW represent monomeric ubiquitin and the polyubiquitinated proteins, respectively. LC-I and LC-II are cytosolic and lipidated forms of LC3, respectively. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and β-tubulin served as controls for TX-soluble and TX-insoluble fractions, respectively.

Article Snippet: Antibodies used for immunohistochemical and immunocytochemical studies included rabbit polyclonal anti-ALS2; HPF1-680 (1∶5,000), rabbit polyclonal anti-SOD1 (1∶500, MBL), guinea pig polyclonal anti-p62/SQSTM1 (1∶1,000, Progen), rabbit polyclonal anti-ubiquitin (1∶200, DakoCytomation), rabbit polyclonal anti-LC3 (1∶1,000, MBL), rabbit polyclonal anti-MAP2 (1∶1,000, CHEMICON), mouse monoclonal anti-GFAP (1∶500, CHEMICON), anti-myelin basic protein (MBP) (1∶5,000, GeneTex), mouse monoclonal anti-EEA1 (1∶100, BD biosciences), mouse monoclonal anti-LAMP2 (1∶250, BD biosciences), and mouse monoclonal anti-FLAG-M2 (1∶500, Stratagene) antibodies.

Techniques: Western Blot, Binding Assay, Molecular Weight

Journal: PLoS ONE

Article Title: Loss of ALS2/Alsin Exacerbates Motor Dysfunction in a SOD1 H46R -Expressing Mouse ALS Model by Disturbing Endolysosomal Trafficking

doi: 10.1371/journal.pone.0009805

Figure Lengend Snippet: ( A ) Representative images of triple immunostaining with MAP2 (green), p62 (red), and GFAP (pink) for the ventral horn of the lumbar spinal cord (L4–L5) from 18-week-old wild-type (WT; 1 st row), 16-week-old Als2 +/+ ; SOD1 H46R (2 nd row), and 16-week-old Als2 −/− ; SOD1 H46R (3 rd row) mice. The nuclei were counterstained with DAPI (Blue). Scale bars = 100 µm. (Lower row) Higher magnification images of p62 immunostaining for the ventral horn of the spinal cord from ( a ) 18-week-old WT, ( b ) 16-week-old Als2 +/+ ; SOD1 H46R , and ( c ) 16-week-old Als2 −/− ; SOD1 H46R mice. It is notable that loss of ALS2 results in an increased number of p62-positive smaller-sized neurons (white arrow) and p62-positive extracellular aggregates (white arrowheads). Scale bars = 20 µm. ( B ) Representative images of triple immunostaining with ubiquitin (Ub) (green), p62 (red), and LC3 (blue) for the ventral horn of the lumbar spinal cord (L4–L5) from 18-week-old WT (upper row), 16-week-old Als2 +/+ ; SOD1 H46R (middle row), and 16-week-old Als2 −/− ; SOD1 H46R (lower row) mice. Red and white arrows represent large motor neurons containing cytoplasmic Ub/p62/LC3-positive puncta and extracellular Ub/p62/LC3-positive aggregates, respectively. The p62-single positive aggregates are also observed (white arrowheads). Scale bars = 10 µm.

Article Snippet: Antibodies used for immunohistochemical and immunocytochemical studies included rabbit polyclonal anti-ALS2; HPF1-680 (1∶5,000), rabbit polyclonal anti-SOD1 (1∶500, MBL), guinea pig polyclonal anti-p62/SQSTM1 (1∶1,000, Progen), rabbit polyclonal anti-ubiquitin (1∶200, DakoCytomation), rabbit polyclonal anti-LC3 (1∶1,000, MBL), rabbit polyclonal anti-MAP2 (1∶1,000, CHEMICON), mouse monoclonal anti-GFAP (1∶500, CHEMICON), anti-myelin basic protein (MBP) (1∶5,000, GeneTex), mouse monoclonal anti-EEA1 (1∶100, BD biosciences), mouse monoclonal anti-LAMP2 (1∶250, BD biosciences), and mouse monoclonal anti-FLAG-M2 (1∶500, Stratagene) antibodies.

Techniques: Triple Immunostaining, Immunostaining

Journal: PLoS ONE

Article Title: Loss of ALS2/Alsin Exacerbates Motor Dysfunction in a SOD1 H46R -Expressing Mouse ALS Model by Disturbing Endolysosomal Trafficking

doi: 10.1371/journal.pone.0009805

Figure Lengend Snippet: Western blot analysis of the levels of proteins, including ALS2, SOD1, ubiquitin (Ub), polyubiquitin binding protein p62/SQSTM1 (p62), microtubule-associated protein 1-light chain 3 (LC3), peripherin, neurofilament heavy chain (NFH), TAR DNA-binding protein 43-kD (TDP-43), heat-shock protein Hsp70, 20S proteasome subunits (α5, C2, and LMP2), vimentin, and glial fibrillary acidic protein (GFAP), in the lumbo-sacral cord from 8, 12, 16, and 20 week-old mice with four distinct genotypes; wild-type ( Als2 +/+ ), Als2 +/+ ; SOD1 H46R , Als2 +/− ; SOD1 H46R , and Als2 −/− ; SOD1 H46R . Two fractions; 1% Triton X-soluble fraction (TX-soluble; left panels) and 1% Triton X-insoluble/5% SDS-soluble fraction (TX-insoluble; right panels) were analyzed. SOD1_mono and SOD1_HMW represent monomeric and high molecular-weight (aggregated) forms of SOD1, respectively. Ub_mono and Ub_HMW represent monomeric ubiquitin and the polyubiquitinated proteins, respectively. LC-I and LC-II are cytosolic and lipidated forms of LC3, respectively. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and β-tubulin served as controls for TX-soluble and TX-insoluble fractions, respectively.

Article Snippet: Antibodies used for immunohistochemical and immunocytochemical studies included rabbit polyclonal anti-ALS2; HPF1-680 (1∶5,000), rabbit polyclonal anti-SOD1 (1∶500, MBL), guinea pig polyclonal anti-p62/SQSTM1 (1∶1,000, Progen), rabbit polyclonal anti-ubiquitin (1∶200, DakoCytomation), rabbit polyclonal anti-LC3 (1∶1,000, MBL), rabbit polyclonal anti-MAP2 (1∶1,000, CHEMICON), mouse monoclonal anti-GFAP (1∶500, CHEMICON), anti-myelin basic protein (MBP) (1∶5,000, GeneTex), mouse monoclonal anti-EEA1 (1∶100, BD biosciences), mouse monoclonal anti-LAMP2 (1∶250, BD biosciences), and mouse monoclonal anti-FLAG-M2 (1∶500, Stratagene) antibodies.

Techniques: Western Blot, Binding Assay, Molecular Weight

Journal: PLoS ONE

Article Title: Loss of ALS2/Alsin Exacerbates Motor Dysfunction in a SOD1 H46R -Expressing Mouse ALS Model by Disturbing Endolysosomal Trafficking

doi: 10.1371/journal.pone.0009805

Figure Lengend Snippet: ( A ) Representative images of triple immunostaining with MAP2 (green), p62 (red), and GFAP (pink) for the ventral horn of the lumbar spinal cord (L4–L5) from 18-week-old wild-type (WT; 1 st row), 16-week-old Als2 +/+ ; SOD1 H46R (2 nd row), and 16-week-old Als2 −/− ; SOD1 H46R (3 rd row) mice. The nuclei were counterstained with DAPI (Blue). Scale bars = 100 µm. (Lower row) Higher magnification images of p62 immunostaining for the ventral horn of the spinal cord from ( a ) 18-week-old WT, ( b ) 16-week-old Als2 +/+ ; SOD1 H46R , and ( c ) 16-week-old Als2 −/− ; SOD1 H46R mice. It is notable that loss of ALS2 results in an increased number of p62-positive smaller-sized neurons (white arrow) and p62-positive extracellular aggregates (white arrowheads). Scale bars = 20 µm. ( B ) Representative images of triple immunostaining with ubiquitin (Ub) (green), p62 (red), and LC3 (blue) for the ventral horn of the lumbar spinal cord (L4–L5) from 18-week-old WT (upper row), 16-week-old Als2 +/+ ; SOD1 H46R (middle row), and 16-week-old Als2 −/− ; SOD1 H46R (lower row) mice. Red and white arrows represent large motor neurons containing cytoplasmic Ub/p62/LC3-positive puncta and extracellular Ub/p62/LC3-positive aggregates, respectively. The p62-single positive aggregates are also observed (white arrowheads). Scale bars = 10 µm.

Article Snippet: Antibodies used for immunohistochemical and immunocytochemical studies included rabbit polyclonal anti-ALS2; HPF1-680 (1∶5,000), rabbit polyclonal anti-SOD1 (1∶500, MBL), guinea pig polyclonal anti-p62/SQSTM1 (1∶1,000, Progen), rabbit polyclonal anti-ubiquitin (1∶200, DakoCytomation), rabbit polyclonal anti-LC3 (1∶1,000, MBL), rabbit polyclonal anti-MAP2 (1∶1,000, CHEMICON), mouse monoclonal anti-GFAP (1∶500, CHEMICON), anti-myelin basic protein (MBP) (1∶5,000, GeneTex), mouse monoclonal anti-EEA1 (1∶100, BD biosciences), mouse monoclonal anti-LAMP2 (1∶250, BD biosciences), and mouse monoclonal anti-FLAG-M2 (1∶500, Stratagene) antibodies.

Techniques: Triple Immunostaining, Immunostaining

Journal: PLoS ONE

Article Title: Loss of ALS2/Alsin Exacerbates Motor Dysfunction in a SOD1 H46R -Expressing Mouse ALS Model by Disturbing Endolysosomal Trafficking

doi: 10.1371/journal.pone.0009805

Figure Lengend Snippet: ( A ) As previously reported , cytoplasmic ALS2 is recruited to membrane ruffles and then to macropinosomes via macropinocytosis upon Rac1 signaling. Subsequently, ALS2 localizing to nascent macropinosomes activates Rab5 and enhances the recruitment of EEA1, PI3K/Vps34, and souble N -ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein complex (not shown), thereby promoting the maturation (fusion and trafficking) of macropinosomes and early endosomes (EE). In the present study, we show that ALS2 is also present to the p62/LC3-positive autophagosomes in cells, suggesting that ALS2 plays a role not only in the maturation of macropinosomes and EE, but also of autophagosomes via their heterotypic fusions, generating amphisomes. The resulting amiphisomes further mature to late endosomes (LE) accompanying the recruitment of Rab7, and ultimately fuse with lysosomes, thereby cargo molecules including poly-ubiquitinated proteins are degraded. Poly-ubiquitinated proteins associated with p62 are also degraded by the proteasomes. ( B ) Loss of ALS2 results in a decrease in endosome fusion, thereby the maturation of autophagosomes and endolysosomal trafficking are disturbed.

Article Snippet: Antibodies used for immunohistochemical and immunocytochemical studies included rabbit polyclonal anti-ALS2; HPF1-680 (1∶5,000), rabbit polyclonal anti-SOD1 (1∶500, MBL), guinea pig polyclonal anti-p62/SQSTM1 (1∶1,000, Progen), rabbit polyclonal anti-ubiquitin (1∶200, DakoCytomation), rabbit polyclonal anti-LC3 (1∶1,000, MBL), rabbit polyclonal anti-MAP2 (1∶1,000, CHEMICON), mouse monoclonal anti-GFAP (1∶500, CHEMICON), anti-myelin basic protein (MBP) (1∶5,000, GeneTex), mouse monoclonal anti-EEA1 (1∶100, BD biosciences), mouse monoclonal anti-LAMP2 (1∶250, BD biosciences), and mouse monoclonal anti-FLAG-M2 (1∶500, Stratagene) antibodies.

Techniques:

Journal: PLoS ONE

Article Title: Loss of ALS2/Alsin Exacerbates Motor Dysfunction in a SOD1 H46R -Expressing Mouse ALS Model by Disturbing Endolysosomal Trafficking

doi: 10.1371/journal.pone.0009805

Figure Lengend Snippet: ( A ) Growth curves for female mice [wild-type (WT) (black circle; n = 14–33), Als2 +/+ ; SOD1 H46R (blue square; n = 6–30), Als2 +/− ; SOD1 H46R (green triangle; n = 28–84), and Als2 −/− ; SOD1 H46R (red inverted triangle; n = 7–44)], and ( B ) for male mice [WT (n = 41–48), Als2 +/+ ; SOD1 H46R (n = 14–46), Als2 +/− ; SOD1 H46R (n = 9–76), and Als2 −/− ; SOD1 H46R (n = 8–36)]. ( A – B ) In either gender, age at which body weight loss began in Als2 −/− ; SOD1 H46R mice was earlier than that for Als2 +/+ ; SOD1 H46R mice (female; *** p <0.001, at 22 weeks, male; *** p <0.001 and ** p <0.01, at 21 and 22 weeks respectively). There were no differences in the mean values between Als2 +/+ ; SOD1 H46R and Als2 +/− ; SOD1 H46R mice at any ages. The values for SOD1 H46R -expressing mice ( Als2 +/+ ; SOD1 H46R , Als2 +/− ; SOD1 H46R , and Als2 −/− ; SOD1 H46R ) later than 8 weeks of ages were all significantly lower than those for WT animals (levels of significance were not shown). Values are mean±SD. Statistical significance is evaluated by ANOVA with Tukey's post hoc test. ( C ) Survival curves for WT [black; n = 55 (female; n = 14, male; n = 41)], Als2 −/− [orange; n = 78 (female; n = 32, male; n = 46)], Als2 +/+ ; SOD1 H46R [blue circle; n = 48 (female; n = 13, male; n = 35)], Als2 +/− ; SOD1 H46R [green square; n = 132 (female; n = 63, male; n = 69)], and Als2 −/− ; SOD1 H46R [red triangle; n = 57 (female; n = 27, male; n = 30)]. Kaplan-Meier analysis identified significant difference between Als2 −/− ; SOD1 H46R and Als2 +/+ ; SOD1 H46R , and between Als2 −/− ; SOD1 H46R and Als2 +/− ; SOD1 H46R (Log-rank test; p <0.0001). ( D ) Survival curves for Als2 +/+ ; SOD1 H46R (blue circle; same as C ), Als2 +/+ ; SOD1 H46R ; ALS2 [gray square; n = 25 (female; n = 13, male; n = 12)], and Als2 −/− ; SOD1 H46R ; ALS2 [pink triangle; n = 11 (female; n = 4, male; n = 7)]. Kaplan-Meier analysis identified no significant differences between groups.

Article Snippet: Antibodies used for immunohistochemical and immunocytochemical studies included rabbit polyclonal anti-ALS2; HPF1-680 (1∶5,000), rabbit polyclonal anti-SOD1 (1∶500, MBL), guinea pig polyclonal anti-p62/SQSTM1 (1∶1,000, Progen), rabbit polyclonal anti-ubiquitin (1∶200, DakoCytomation), rabbit polyclonal anti-LC3 (1∶1,000, MBL), rabbit polyclonal anti-MAP2 (1∶1,000, CHEMICON), mouse monoclonal anti-GFAP (1∶500, CHEMICON), anti-myelin basic protein (MBP) (1∶5,000, GeneTex), mouse monoclonal anti-EEA1 (1∶100, BD biosciences), mouse monoclonal anti-LAMP2 (1∶250, BD biosciences), and mouse monoclonal anti-FLAG-M2 (1∶500, Stratagene) antibodies.

Techniques: Expressing

Journal: PLoS ONE

Article Title: Loss of ALS2/Alsin Exacerbates Motor Dysfunction in a SOD1 H46R -Expressing Mouse ALS Model by Disturbing Endolysosomal Trafficking

doi: 10.1371/journal.pone.0009805

Figure Lengend Snippet: ( A ) Changes in the balance beam test scores in wild-type (WT) (black circle), Als2 −/− (orange square), Als2 +/+ ; SOD1 H46R (blue triangle), Als2 +/− ; SOD1 H46R (green inverted triangle), and Als2 −/− ; SOD1 H46R (red diamond) mice. Values are means±SEM [each genotype; n = 20 (female; n = 10, male; n = 10)]. Statistical significance is evaluated by ANOVA with Scheffé's post hoc test. There are significant differences between Als2 +/+ ; SOD1 H46R (blue) and Als2 −/− ; SOD1 H46R (red) mice (* p <0.05, ** p <0.01, or *** p <0.001 at 15–21 weeks of ages), and between Als2 +/+ ; SOD1 H46R (blue) and Als2 +/− ; SOD1 H46R (green) mice (++ p <0.01 at 21 weeks of age). ( B ) The rearing and ( C ) cage activities in wild-type ( Als2 +/+ ), Als2 +/− ; SOD1 H46R , and Als2 −/− ; SOD1 H46R mice in a dark (gray) and a light (white) cycle at 12 and 18 weeks of ages. Cumulative data counting for 7 consecutive days are shown as Box-Wisker plots [AU; arbitrary unit, each genotype; n = 8–10 (female)]. Statistical significance is evaluated by non-parametric ANOVA (Kruskal-Wallis) with Dunn's post hoc test. There are significant differences in the rearing activities (dark & light) between wild-type ( Als2 +/+ ) and Als2 −/− ; SOD1 H46R mice (*** p <0.01), in the rearing activities (light) between Als2 +/− ; SOD1 H46R and Als2 −/− ; SOD1 H46R mice (*** p <0.05), and in the cage activities between wild-type ( Als2 +/+ ) and Als2 −/− ; SOD1 H46R mice (*** p <0.01) at 18 weeks of age.

Article Snippet: Antibodies used for immunohistochemical and immunocytochemical studies included rabbit polyclonal anti-ALS2; HPF1-680 (1∶5,000), rabbit polyclonal anti-SOD1 (1∶500, MBL), guinea pig polyclonal anti-p62/SQSTM1 (1∶1,000, Progen), rabbit polyclonal anti-ubiquitin (1∶200, DakoCytomation), rabbit polyclonal anti-LC3 (1∶1,000, MBL), rabbit polyclonal anti-MAP2 (1∶1,000, CHEMICON), mouse monoclonal anti-GFAP (1∶500, CHEMICON), anti-myelin basic protein (MBP) (1∶5,000, GeneTex), mouse monoclonal anti-EEA1 (1∶100, BD biosciences), mouse monoclonal anti-LAMP2 (1∶250, BD biosciences), and mouse monoclonal anti-FLAG-M2 (1∶500, Stratagene) antibodies.

Techniques:

Journal: PLoS ONE

Article Title: Loss of ALS2/Alsin Exacerbates Motor Dysfunction in a SOD1 H46R -Expressing Mouse ALS Model by Disturbing Endolysosomal Trafficking

doi: 10.1371/journal.pone.0009805

Figure Lengend Snippet: ( A ) Representative toluidine blue staining of the transverse section of lumbar spinal cord (L4–L5) from 18-week-old wild-type (WT; upper row), 16-week-old Als2 +/+ ; SOD1 H46R (middle row), and 16-week-old Als2 −/− ; SOD1 H46R (lower row) mice. Images for the dorsal columns, lateral columns, ventral columns, and ventral horn cells were shown. Red arrowheads indicate degenerating axons. Axonal degeneration is most prominent in Als2 −/− ; SOD1 H46R mice. Scale bars = 20 µm. ( B – F ) Representative electron micrographs of lumbar (L4–L5) spinal axons from Als2 −/− ; SOD1 H46R mouse at 16 weeks ( B – F ) and 8 weeks ( G ). Degenerating axon ( B ), axon accumulating fibrillar materials and multivesicular bodies ( C and c′ ), membrane saccule containing granular/osmiophilic aggregates and autophagosome-like vesicles ( D and d′ , red arrowhead), axon containing osmiophilic and autophagosome-like (red arrowhead) vesicles ( E and e′ ), astrocyte containing osmiophilic aggregates ( F ), and swollen axon accumulating granular aggregates and vesicles ( G ), are shown. Scale bars are as indicated.

Article Snippet: Antibodies used for immunohistochemical and immunocytochemical studies included rabbit polyclonal anti-ALS2; HPF1-680 (1∶5,000), rabbit polyclonal anti-SOD1 (1∶500, MBL), guinea pig polyclonal anti-p62/SQSTM1 (1∶1,000, Progen), rabbit polyclonal anti-ubiquitin (1∶200, DakoCytomation), rabbit polyclonal anti-LC3 (1∶1,000, MBL), rabbit polyclonal anti-MAP2 (1∶1,000, CHEMICON), mouse monoclonal anti-GFAP (1∶500, CHEMICON), anti-myelin basic protein (MBP) (1∶5,000, GeneTex), mouse monoclonal anti-EEA1 (1∶100, BD biosciences), mouse monoclonal anti-LAMP2 (1∶250, BD biosciences), and mouse monoclonal anti-FLAG-M2 (1∶500, Stratagene) antibodies.

Techniques: Staining

Journal: PLoS ONE

Article Title: Loss of ALS2/Alsin Exacerbates Motor Dysfunction in a SOD1 H46R -Expressing Mouse ALS Model by Disturbing Endolysosomal Trafficking

doi: 10.1371/journal.pone.0009805

Figure Lengend Snippet: Western blot analysis of the levels of proteins, including ALS2, SOD1, ubiquitin (Ub), polyubiquitin binding protein p62/SQSTM1 (p62), microtubule-associated protein 1-light chain 3 (LC3), peripherin, neurofilament heavy chain (NFH), TAR DNA-binding protein 43-kD (TDP-43), heat-shock protein Hsp70, 20S proteasome subunits (α5, C2, and LMP2), vimentin, and glial fibrillary acidic protein (GFAP), in the lumbo-sacral cord from 8, 12, 16, and 20 week-old mice with four distinct genotypes; wild-type ( Als2 +/+ ), Als2 +/+ ; SOD1 H46R , Als2 +/− ; SOD1 H46R , and Als2 −/− ; SOD1 H46R . Two fractions; 1% Triton X-soluble fraction (TX-soluble; left panels) and 1% Triton X-insoluble/5% SDS-soluble fraction (TX-insoluble; right panels) were analyzed. SOD1_mono and SOD1_HMW represent monomeric and high molecular-weight (aggregated) forms of SOD1, respectively. Ub_mono and Ub_HMW represent monomeric ubiquitin and the polyubiquitinated proteins, respectively. LC-I and LC-II are cytosolic and lipidated forms of LC3, respectively. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and β-tubulin served as controls for TX-soluble and TX-insoluble fractions, respectively.

Article Snippet: Antibodies used for immunohistochemical and immunocytochemical studies included rabbit polyclonal anti-ALS2; HPF1-680 (1∶5,000), rabbit polyclonal anti-SOD1 (1∶500, MBL), guinea pig polyclonal anti-p62/SQSTM1 (1∶1,000, Progen), rabbit polyclonal anti-ubiquitin (1∶200, DakoCytomation), rabbit polyclonal anti-LC3 (1∶1,000, MBL), rabbit polyclonal anti-MAP2 (1∶1,000, CHEMICON), mouse monoclonal anti-GFAP (1∶500, CHEMICON), anti-myelin basic protein (MBP) (1∶5,000, GeneTex), mouse monoclonal anti-EEA1 (1∶100, BD biosciences), mouse monoclonal anti-LAMP2 (1∶250, BD biosciences), and mouse monoclonal anti-FLAG-M2 (1∶500, Stratagene) antibodies.

Techniques: Western Blot, Binding Assay, Molecular Weight

Journal: PLoS ONE

Article Title: Loss of ALS2/Alsin Exacerbates Motor Dysfunction in a SOD1 H46R -Expressing Mouse ALS Model by Disturbing Endolysosomal Trafficking

doi: 10.1371/journal.pone.0009805

Figure Lengend Snippet: Chymotrypsin-like activity in the lumbo-sacral cord from 18, 20, and 23 week-old mice with four distinct genotypes; wild-type (WT), Als2 −/− , Als2 +/+ ; SOD1 H46R , and Als2 −/− ; SOD1 H46R are measured. Values are mean ± SD (n = 3–5) in percent (%) relative to 18 week-old wild-type mice. Statistical significance is evaluated by ANOVA with Bonferroni's post hoc test (* p <0.05, *** p <0.001).

Article Snippet: Antibodies used for immunohistochemical and immunocytochemical studies included rabbit polyclonal anti-ALS2; HPF1-680 (1∶5,000), rabbit polyclonal anti-SOD1 (1∶500, MBL), guinea pig polyclonal anti-p62/SQSTM1 (1∶1,000, Progen), rabbit polyclonal anti-ubiquitin (1∶200, DakoCytomation), rabbit polyclonal anti-LC3 (1∶1,000, MBL), rabbit polyclonal anti-MAP2 (1∶1,000, CHEMICON), mouse monoclonal anti-GFAP (1∶500, CHEMICON), anti-myelin basic protein (MBP) (1∶5,000, GeneTex), mouse monoclonal anti-EEA1 (1∶100, BD biosciences), mouse monoclonal anti-LAMP2 (1∶250, BD biosciences), and mouse monoclonal anti-FLAG-M2 (1∶500, Stratagene) antibodies.

Techniques: Activity Assay

Journal: PLoS ONE

Article Title: Loss of ALS2/Alsin Exacerbates Motor Dysfunction in a SOD1 H46R -Expressing Mouse ALS Model by Disturbing Endolysosomal Trafficking

doi: 10.1371/journal.pone.0009805

Figure Lengend Snippet: ( A ) Representative images of triple immunostaining with MAP2 (green), p62 (red), and GFAP (pink) for the ventral horn of the lumbar spinal cord (L4–L5) from 18-week-old wild-type (WT; 1 st row), 16-week-old Als2 +/+ ; SOD1 H46R (2 nd row), and 16-week-old Als2 −/− ; SOD1 H46R (3 rd row) mice. The nuclei were counterstained with DAPI (Blue). Scale bars = 100 µm. (Lower row) Higher magnification images of p62 immunostaining for the ventral horn of the spinal cord from ( a ) 18-week-old WT, ( b ) 16-week-old Als2 +/+ ; SOD1 H46R , and ( c ) 16-week-old Als2 −/− ; SOD1 H46R mice. It is notable that loss of ALS2 results in an increased number of p62-positive smaller-sized neurons (white arrow) and p62-positive extracellular aggregates (white arrowheads). Scale bars = 20 µm. ( B ) Representative images of triple immunostaining with ubiquitin (Ub) (green), p62 (red), and LC3 (blue) for the ventral horn of the lumbar spinal cord (L4–L5) from 18-week-old WT (upper row), 16-week-old Als2 +/+ ; SOD1 H46R (middle row), and 16-week-old Als2 −/− ; SOD1 H46R (lower row) mice. Red and white arrows represent large motor neurons containing cytoplasmic Ub/p62/LC3-positive puncta and extracellular Ub/p62/LC3-positive aggregates, respectively. The p62-single positive aggregates are also observed (white arrowheads). Scale bars = 10 µm.

Article Snippet: Antibodies used for immunohistochemical and immunocytochemical studies included rabbit polyclonal anti-ALS2; HPF1-680 (1∶5,000), rabbit polyclonal anti-SOD1 (1∶500, MBL), guinea pig polyclonal anti-p62/SQSTM1 (1∶1,000, Progen), rabbit polyclonal anti-ubiquitin (1∶200, DakoCytomation), rabbit polyclonal anti-LC3 (1∶1,000, MBL), rabbit polyclonal anti-MAP2 (1∶1,000, CHEMICON), mouse monoclonal anti-GFAP (1∶500, CHEMICON), anti-myelin basic protein (MBP) (1∶5,000, GeneTex), mouse monoclonal anti-EEA1 (1∶100, BD biosciences), mouse monoclonal anti-LAMP2 (1∶250, BD biosciences), and mouse monoclonal anti-FLAG-M2 (1∶500, Stratagene) antibodies.

Techniques: Triple Immunostaining, Immunostaining

Journal: PLoS ONE

Article Title: Loss of ALS2/Alsin Exacerbates Motor Dysfunction in a SOD1 H46R -Expressing Mouse ALS Model by Disturbing Endolysosomal Trafficking

doi: 10.1371/journal.pone.0009805

Figure Lengend Snippet: ( A ) Ectopically expressed EGFP-LC3 (LC3), FLAG-tagged SOD1 (SOD1: SOD1 WT ; WT, SOD1 H46R ; H46R, SOD1 G85R ; G85R, SOD1 G93A ; G93A, SOD1 A4V ; A4V), and ALS2 were diffusedly distributed throughout the cytosol with no colocalization in NSC-34 cells under normal conditions (Control). ( B ) Under the treatment with 50 µM chloroquine for 18 hr, by which endolysosomal protein degradation was severely inhibited, ectopically expressed SOD1 WT , SOD1 H46R , and SOD1 G85R were partially colocalized with LC3/ALS2 onto enlarged endolysosomal vesicular compartments in NSC-34 cells (white arrows in enlarged images). Fourth and fifth columns display the merged images for double (LC3 and SOD1) and triple stainings, respectively ( A and B ). Sixth columns in B (Enlarge) represent a higher magnification of the merged-images of the respective 5 th columns in B . Scale bars = 10 µm.

Article Snippet: Antibodies used for immunohistochemical and immunocytochemical studies included rabbit polyclonal anti-ALS2; HPF1-680 (1∶5,000), rabbit polyclonal anti-SOD1 (1∶500, MBL), guinea pig polyclonal anti-p62/SQSTM1 (1∶1,000, Progen), rabbit polyclonal anti-ubiquitin (1∶200, DakoCytomation), rabbit polyclonal anti-LC3 (1∶1,000, MBL), rabbit polyclonal anti-MAP2 (1∶1,000, CHEMICON), mouse monoclonal anti-GFAP (1∶500, CHEMICON), anti-myelin basic protein (MBP) (1∶5,000, GeneTex), mouse monoclonal anti-EEA1 (1∶100, BD biosciences), mouse monoclonal anti-LAMP2 (1∶250, BD biosciences), and mouse monoclonal anti-FLAG-M2 (1∶500, Stratagene) antibodies.

Techniques:

Journal: PLoS ONE

Article Title: Loss of ALS2/Alsin Exacerbates Motor Dysfunction in a SOD1 H46R -Expressing Mouse ALS Model by Disturbing Endolysosomal Trafficking

doi: 10.1371/journal.pone.0009805

Figure Lengend Snippet: Western blot analysis of the levels of proteins, including ALS2, SOD1, ubiquitin (Ub), polyubiquitin binding protein p62/SQSTM1 (p62), microtubule-associated protein 1-light chain 3 (LC3), peripherin, neurofilament heavy chain (NFH), TAR DNA-binding protein 43-kD (TDP-43), heat-shock protein Hsp70, 20S proteasome subunits (α5, C2, and LMP2), vimentin, and glial fibrillary acidic protein (GFAP), in the lumbo-sacral cord from 8, 12, 16, and 20 week-old mice with four distinct genotypes; wild-type ( Als2 +/+ ), Als2 +/+ ; SOD1 H46R , Als2 +/− ; SOD1 H46R , and Als2 −/− ; SOD1 H46R . Two fractions; 1% Triton X-soluble fraction (TX-soluble; left panels) and 1% Triton X-insoluble/5% SDS-soluble fraction (TX-insoluble; right panels) were analyzed. SOD1_mono and SOD1_HMW represent monomeric and high molecular-weight (aggregated) forms of SOD1, respectively. Ub_mono and Ub_HMW represent monomeric ubiquitin and the polyubiquitinated proteins, respectively. LC-I and LC-II are cytosolic and lipidated forms of LC3, respectively. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and β-tubulin served as controls for TX-soluble and TX-insoluble fractions, respectively.

Article Snippet: Antibodies used for immunohistochemical and immunocytochemical studies included rabbit polyclonal anti-ALS2; HPF1-680 (1∶5,000), rabbit polyclonal anti-SOD1 (1∶500, MBL), guinea pig polyclonal anti-p62/SQSTM1 (1∶1,000, Progen), rabbit polyclonal anti-ubiquitin (1∶200, DakoCytomation), rabbit polyclonal anti-LC3 (1∶1,000, MBL), rabbit polyclonal anti-MAP2 (1∶1,000, CHEMICON), mouse monoclonal anti-GFAP (1∶500, CHEMICON), anti-myelin basic protein (MBP) (1∶5,000, GeneTex), mouse monoclonal anti-EEA1 (1∶100, BD biosciences), mouse monoclonal anti-LAMP2 (1∶250, BD biosciences), and mouse monoclonal anti-FLAG-M2 (1∶500, Stratagene) antibodies.

Techniques: Western Blot, Binding Assay, Molecular Weight

Journal: PLoS ONE

Article Title: Loss of ALS2/Alsin Exacerbates Motor Dysfunction in a SOD1 H46R -Expressing Mouse ALS Model by Disturbing Endolysosomal Trafficking

doi: 10.1371/journal.pone.0009805

Figure Lengend Snippet: ( A ) Representative images of triple immunostaining with MAP2 (green), p62 (red), and GFAP (pink) for the ventral horn of the lumbar spinal cord (L4–L5) from 18-week-old wild-type (WT; 1 st row), 16-week-old Als2 +/+ ; SOD1 H46R (2 nd row), and 16-week-old Als2 −/− ; SOD1 H46R (3 rd row) mice. The nuclei were counterstained with DAPI (Blue). Scale bars = 100 µm. (Lower row) Higher magnification images of p62 immunostaining for the ventral horn of the spinal cord from ( a ) 18-week-old WT, ( b ) 16-week-old Als2 +/+ ; SOD1 H46R , and ( c ) 16-week-old Als2 −/− ; SOD1 H46R mice. It is notable that loss of ALS2 results in an increased number of p62-positive smaller-sized neurons (white arrow) and p62-positive extracellular aggregates (white arrowheads). Scale bars = 20 µm. ( B ) Representative images of triple immunostaining with ubiquitin (Ub) (green), p62 (red), and LC3 (blue) for the ventral horn of the lumbar spinal cord (L4–L5) from 18-week-old WT (upper row), 16-week-old Als2 +/+ ; SOD1 H46R (middle row), and 16-week-old Als2 −/− ; SOD1 H46R (lower row) mice. Red and white arrows represent large motor neurons containing cytoplasmic Ub/p62/LC3-positive puncta and extracellular Ub/p62/LC3-positive aggregates, respectively. The p62-single positive aggregates are also observed (white arrowheads). Scale bars = 10 µm.

Article Snippet: Antibodies used for immunohistochemical and immunocytochemical studies included rabbit polyclonal anti-ALS2; HPF1-680 (1∶5,000), rabbit polyclonal anti-SOD1 (1∶500, MBL), guinea pig polyclonal anti-p62/SQSTM1 (1∶1,000, Progen), rabbit polyclonal anti-ubiquitin (1∶200, DakoCytomation), rabbit polyclonal anti-LC3 (1∶1,000, MBL), rabbit polyclonal anti-MAP2 (1∶1,000, CHEMICON), mouse monoclonal anti-GFAP (1∶500, CHEMICON), anti-myelin basic protein (MBP) (1∶5,000, GeneTex), mouse monoclonal anti-EEA1 (1∶100, BD biosciences), mouse monoclonal anti-LAMP2 (1∶250, BD biosciences), and mouse monoclonal anti-FLAG-M2 (1∶500, Stratagene) antibodies.

Techniques: Triple Immunostaining, Immunostaining

Journal: PLoS ONE

Article Title: Loss of ALS2/Alsin Exacerbates Motor Dysfunction in a SOD1 H46R -Expressing Mouse ALS Model by Disturbing Endolysosomal Trafficking

doi: 10.1371/journal.pone.0009805

Figure Lengend Snippet: Western blot analysis of the levels of proteins, including ALS2, SOD1, ubiquitin (Ub), polyubiquitin binding protein p62/SQSTM1 (p62), microtubule-associated protein 1-light chain 3 (LC3), peripherin, neurofilament heavy chain (NFH), TAR DNA-binding protein 43-kD (TDP-43), heat-shock protein Hsp70, 20S proteasome subunits (α5, C2, and LMP2), vimentin, and glial fibrillary acidic protein (GFAP), in the lumbo-sacral cord from 8, 12, 16, and 20 week-old mice with four distinct genotypes; wild-type ( Als2 +/+ ), Als2 +/+ ; SOD1 H46R , Als2 +/− ; SOD1 H46R , and Als2 −/− ; SOD1 H46R . Two fractions; 1% Triton X-soluble fraction (TX-soluble; left panels) and 1% Triton X-insoluble/5% SDS-soluble fraction (TX-insoluble; right panels) were analyzed. SOD1_mono and SOD1_HMW represent monomeric and high molecular-weight (aggregated) forms of SOD1, respectively. Ub_mono and Ub_HMW represent monomeric ubiquitin and the polyubiquitinated proteins, respectively. LC-I and LC-II are cytosolic and lipidated forms of LC3, respectively. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and β-tubulin served as controls for TX-soluble and TX-insoluble fractions, respectively.

Article Snippet: Primary antibodies used for western blot analysis included two independent rabbit polyclonal anti-ALS2; HPF1-680 (1∶3,000) and MPF1012-1651 (1∶3,000) , rabbit polyclonal anti-SOD1 (FL-154) (1∶20,000, Santa Cruz), mouse monoclonal anti-ubiquitin (P4D1) (1∶3,000, Santa Cruz), guinea pig polyclonal anti-p62/SQSTM1 (1∶6,000, Progen), rabbit polyclonal anti-LC3 (1∶5,000, MBL), rabbit polyclonal anti-peripherin (1∶5,000, CHEMICON), mouse monoclonal anti-NFH (1∶12,000, Sigma), rabbit polyclonal anti-TDP-43 (1∶2,000, Protein Tech Group), mouse monoclonal anti-Hsp70 (1∶3,000, Santa Cruz), rabbit polyclonal anti-proteasome 20S subunit alpha-5 (1∶3,000, Thermo Scientific), rabbit polyclonal anti-proteasome 20S C2 (1∶3,000, Thermo Scientific), rabbit polyclonal anti-proteasome 20S LMP2 (Novus), mouse monoclonal anti-vimentin (1∶3,000, Sigma), rabbit polyclonal anti-GFAP (1∶50,000, Biomeda), mouse monoclonal anti-β-tubulin (1∶100,000, CHEMICON), mouse monoclonal anti-α-tubulin (1∶10,000, Sigma), and mouse monoclonal anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1∶10,000, CHEMICON) antibodies.

Techniques: Western Blot, Binding Assay, Molecular Weight

Journal: PLoS ONE

Article Title: Loss of ALS2/Alsin Exacerbates Motor Dysfunction in a SOD1 H46R -Expressing Mouse ALS Model by Disturbing Endolysosomal Trafficking

doi: 10.1371/journal.pone.0009805

Figure Lengend Snippet: Western blot analysis of the levels of proteins, including ALS2, SOD1, ubiquitin (Ub), polyubiquitin binding protein p62/SQSTM1 (p62), microtubule-associated protein 1-light chain 3 (LC3), peripherin, neurofilament heavy chain (NFH), TAR DNA-binding protein 43-kD (TDP-43), heat-shock protein Hsp70, 20S proteasome subunits (α5, C2, and LMP2), vimentin, and glial fibrillary acidic protein (GFAP), in the lumbo-sacral cord from 8, 12, 16, and 20 week-old mice with four distinct genotypes; wild-type ( Als2 +/+ ), Als2 +/+ ; SOD1 H46R , Als2 +/− ; SOD1 H46R , and Als2 −/− ; SOD1 H46R . Two fractions; 1% Triton X-soluble fraction (TX-soluble; left panels) and 1% Triton X-insoluble/5% SDS-soluble fraction (TX-insoluble; right panels) were analyzed. SOD1_mono and SOD1_HMW represent monomeric and high molecular-weight (aggregated) forms of SOD1, respectively. Ub_mono and Ub_HMW represent monomeric ubiquitin and the polyubiquitinated proteins, respectively. LC-I and LC-II are cytosolic and lipidated forms of LC3, respectively. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and β-tubulin served as controls for TX-soluble and TX-insoluble fractions, respectively.

Article Snippet: Primary antibodies used for western blot analysis included two independent rabbit polyclonal anti-ALS2; HPF1-680 (1∶3,000) and MPF1012-1651 (1∶3,000) , rabbit polyclonal anti-SOD1 (FL-154) (1∶20,000, Santa Cruz), mouse monoclonal anti-ubiquitin (P4D1) (1∶3,000, Santa Cruz), guinea pig polyclonal anti-p62/SQSTM1 (1∶6,000, Progen), rabbit polyclonal anti-LC3 (1∶5,000, MBL), rabbit polyclonal anti-peripherin (1∶5,000, CHEMICON), mouse monoclonal anti-NFH (1∶12,000, Sigma), rabbit polyclonal anti-TDP-43 (1∶2,000, Protein Tech Group), mouse monoclonal anti-Hsp70 (1∶3,000, Santa Cruz), rabbit polyclonal anti-proteasome 20S subunit alpha-5 (1∶3,000, Thermo Scientific), rabbit polyclonal anti-proteasome 20S C2 (1∶3,000, Thermo Scientific), rabbit polyclonal anti-proteasome 20S LMP2 (Novus), mouse monoclonal anti-vimentin (1∶3,000, Sigma), rabbit polyclonal anti-GFAP (1∶50,000, Biomeda), mouse monoclonal anti-β-tubulin (1∶100,000, CHEMICON), mouse monoclonal anti-α-tubulin (1∶10,000, Sigma), and mouse monoclonal anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1∶10,000, CHEMICON) antibodies.

Techniques: Western Blot, Binding Assay, Molecular Weight

Journal: Developmental cell

Article Title: Endolysosomal Targeting of Mitochondria is Integral to BAX-mediated Mitochondrial Permeabilization During Apoptosis Signaling

doi: 10.1016/j.devcel.2020.05.014

Figure Lengend Snippet: (A) MCF-7_Cyto c-GFP cells, in normal growth medium (full medium, FM) or treated with STS (1 μM) for 4 hrs, TNF α/AcD (50 ng/mL / 1 μg/mL) for 6 hrs, or CPT (20 μM) for 24 hrs. IF of Rab5 and TOM70. Scale bars, 10 μm.

Article Snippet: Cells were then incubated with primary antibodies against BAX (Santa Cruz Biotechnology; ABclonal), cytochrome c (BD Biosciences), Rabex-5 (Santa Cruz Biotechnology), Smac/DIABLO (Santa Cruz Biotechnology), TOM20 (Santa Cruz Biotechnology), TOM70 (Santa Cruz Biotechnology), at room temperature for 1 hr, or with Rab5 (Cell Signaling) or ALS2 (Sigma-Aldrich; ABclonal) antibodies at 4°C overnight.

Techniques:

Journal: Developmental cell

Article Title: Endolysosomal Targeting of Mitochondria is Integral to BAX-mediated Mitochondrial Permeabilization During Apoptosis Signaling

doi: 10.1016/j.devcel.2020.05.014

Figure Lengend Snippet: (A) MCF-7_Cyto c-GFP_RFP-Rab5 cells, live imaged every 5 sec over 2 min under FM conditions. Left, merged channel single time-point images with EL-mitochondrial interactions overlaid in white. Right, color-coded EL-mitochondrial interactions, projected over 2 min.

Article Snippet: Cells were then incubated with primary antibodies against BAX (Santa Cruz Biotechnology; ABclonal), cytochrome c (BD Biosciences), Rabex-5 (Santa Cruz Biotechnology), Smac/DIABLO (Santa Cruz Biotechnology), TOM20 (Santa Cruz Biotechnology), TOM70 (Santa Cruz Biotechnology), at room temperature for 1 hr, or with Rab5 (Cell Signaling) or ALS2 (Sigma-Aldrich; ABclonal) antibodies at 4°C overnight.

Techniques:

Journal: Developmental cell

Article Title: Endolysosomal Targeting of Mitochondria is Integral to BAX-mediated Mitochondrial Permeabilization During Apoptosis Signaling

doi: 10.1016/j.devcel.2020.05.014

Figure Lengend Snippet: (A) Immunoblot of Rab5 levels in MCF-7 cells stably expressing shCON, shRab5A or shRab5C. GAPDH, loading control.

Article Snippet: Cells were then incubated with primary antibodies against BAX (Santa Cruz Biotechnology; ABclonal), cytochrome c (BD Biosciences), Rabex-5 (Santa Cruz Biotechnology), Smac/DIABLO (Santa Cruz Biotechnology), TOM20 (Santa Cruz Biotechnology), TOM70 (Santa Cruz Biotechnology), at room temperature for 1 hr, or with Rab5 (Cell Signaling) or ALS2 (Sigma-Aldrich; ABclonal) antibodies at 4°C overnight.

Techniques: Western Blot, Stable Transfection, Expressing