Journal: Scientific Reports

Article Title: Syntabulin regulates the trafficking of PICK1-containing vesicles in neurons

doi: 10.1038/srep20924

Figure Lengend Snippet: ( A ) Neurons were infected with syntabulin shRNA lentivirus as indicated at DIV5 and examined at DIV13. Syntabulin knockdown significantly increased total ASIC2 expression but reduced surface ASIC2 levels as compared with the levels in control and scrambled-shRNA control. ( B ) Quantification of normalized ASIC2/GAPDH ratio, total ASIC2/surface ASIC2 ratio and surface ASIC2 levels. One-way ANOVA and Sidak test; ** P = 0.008, and P = 0.009 for Stb sh#1 Total ASIC2/GAPDH and Surface ASIC2/GAPDH compared to control respectively; * P = 0.044 and * P = 0.016 for Stb #1 total ASIC2/GAPDH and Surface/Total ASIC2 ratio compared to scrambled shRNA control respectively; * P = 0.020 for Stb #1 Surface/Total ASIC2 ratio compared to control; “ns” P > 0.05. n = 4 − 6 independent experiments. Error bars represent the SEM. ( C , D ) Hoechst staining of neurons after syntabulin knockdown, in pH 7.4 solution and pH6.0 solution. Knockdown of syntabulin significantly increased cell death as compared with controls in pH6.0. Scale bar = 20 μm. ( E ) Quantification of normalized total Hoechst intensity. One-way ANOVA and Sidak test; * P = 0.035 compared to control; * P = 0.043 compared to scrambled-shRNA control; “ns” P > 0.05; n = 6 independent experimental repeats. Error bars represent the SEM.

Article Snippet: The following antibodies were purchased: rabbit polyclonal ASIC2 antibody, Proteintech (17851-1-AP); mouse myc and β-tubulin antibodies, DSHB (9E10 and E7); mouse GAPDH antibody, Beyotime (GA019); mouse Map2 antibody, Sigma (M9942); and mouse β-actin antibody, Sigma (A5316).

Techniques: Infection, shRNA, Knockdown, Expressing, Control, Staining

Journal: The Journal of Neuroscience

Article Title: Acid-Sensing Ion Channel 2 Is Important for Retinal Function and Protects against Light-Induced Retinal Degeneration

doi: 10.1523/JNEUROSCI.4698-03.2004

Figure Lengend Snippet: ASIC transcripts and protein in the mouse retina. A–D, In situ hybridization with retinas from 3-month-old ASIC2+/+ (A, C) and ASIC2–/– (B, D) mice with ASIC2a (A, B) or ASIC2b (C, D) probes. In wild-type mice, ASIC2a and ASIC2b mRNAs are expressed in the outer nuclear layer (ONL) and in some cells of the distal and proximal portion of the inner nuclear layer (INL; arrows). Expression of both transcripts was reduced in ASIC2 null mice. Similar results were obtained with three antisense oligonucleotides for each transcript. Both omission of the oligonucleotide and incubation with a DIG-labeled oligonucleotide unrelated to ASIC2 did not yield any labeling. OS, Outer segment; IS, inner segment; OPL, outer plexiform layer; IPL, inner plexiform layer; GCL, ganglion cell layer. E, Western blot with an antibody directed against the ASIC2a NH2 terminus confirmed the presence of ASIC2a in the retina. COS, A total of 6 μg of homogenate from ASIC2a-transfected COS cells. +/+ and –/–, A total of 20 μg of retina homogenate from ASIC2+/+ or ASIC2–/– mice. Although a protein of ∼60 kDa is labeled in ASIC2+/+ mice, neither the 60 kDa protein nor the 7 kDa shorter protein for which the targeted ASIC2 transcript codes is detected in ASIC2–/– mice. The smaller apparent molecular weight of ASIC2 heterologously expressed in COS is probably attributable to a lower glycosylation level. The proteins detected on the blot are recognized by the anti-ASIC2 antibody, because omission of the primary antibody abolished their labeling (data not shown). The anti-ASIC2 antibodies that we prepared as well as the commercialized anti ASIC2a-antibodies obtained from Alomone Labs, Chemicon, and Alpha Diagnostics directed against NH2 terminal peptides all cross-react strongly with another protein slightly bigger than ASIC2a in ASIC2–/– mice.

Article Snippet: The anti-ASIC2 antibodies that we prepared as well as the commercialized anti ASIC2a-antibodies obtained from Alomone Labs, Chemicon, and Alpha Diagnostics directed against NH2 terminal peptides all cross-react strongly with another protein slightly bigger than ASIC2a in ASIC2 –/– mice.

Techniques: In Situ Hybridization, Expressing, Incubation, Labeling, Western Blot, Transfection, Molecular Weight

Journal: Journal of Cellular and Molecular Medicine

Article Title: Acid‐sensing ion channel 1a is involved in ischaemia/reperfusion induced kidney injury by increasing renal epithelia cell apoptosis

doi: 10.1111/jcmm.14238

Figure Lengend Snippet: Immunohistochemical analysis of ASIC1a expression in renal tissue. Immunostaining for ASIC1a, AQP1, THP and SYN in renal tissue. ASIC1a was colocalized with AQP and SYN, but not THP. ASIC1a, acid sensing ion channel 1a; AQP1, aquaporin 1, proximal tubular cells marker; THP, Tamm‐Horsfall protein, thick ascending limb and distal tubular cells marker; SYN, Synaptopodin, podocyte marker; DAPI, 4,6‐diamidino‐2‐phenylindole, nuclear

Article Snippet: After incubated in 0.3% BSA, the slices was incubated in the mixed primary antibodies: Guinea pig anti‐ASIC1a (1:50 alomone lab, Israel)+Rabbit anti‐aquaporin 1 (AQP1, 1:100, Abcam, UAS), Guinea pig anti‐ASIC1a (1:50)+Rabbit anti‐ Tamm‐Horsfall protein (THP, 1:100, Abcam, UAS), Guinea pig anti‐ASIC1a (1:50)+Rabbit anti‐Synaptopodin (SYN, 1:100, Abcam, USA), Guinea pig anti‐ASIC2a (1:50 alomone lab, Israel)+Rabbit anti‐AQP1(1:100), Guinea pig anti‐ASIC2a (1:50)+Rabbit anti‐THP(1:100), Guinea pig anti‐ASIC2a (1:50)+Rabbit anti‐SYN (1:100), Guinea pig anti‐ASIC3 (1:50 alomone lab, Israel)+Rabbit anti‐AQP1(1:100), Guinea pig anti‐ASIC3 (1:50)+Rabbit anti‐THP(1:100), Guinea pig anti‐ASIC3 (1:50)+Rabbit anti‐ SYN (1:100).

Techniques: Immunohistochemical staining, Expressing, Immunostaining, Marker

Journal: Neural Regeneration Research

Article Title: Ischemic postconditioning protects against ischemic brain injury by up-regulation of acid-sensing ion channel 2a

doi: 10.4103/1673-5374.180751

Figure Lengend Snippet: ASIC2a protein expression in the rat hippocampal CA1 region. Ischemic postconditioning strongly up-regulated ASIC2a protein expression in the rat hippocampal CA1 region after ischemic brain injury. ASIC2a: Acid-sensing ion channel 2a; GAPDH: glyceraldehye phosphate dehydrogenase.

Article Snippet: Membranes were blocked using 5% skimmed milk, incubated at room temperature for 120 minutes, washed four times (10 minutes each) in PBS-Tween, incubated with primary rabbit anti-rat ASIC2a and GAPDH polyclonal antibodies (1:5,000; Sigma, St Louis, MO, USA) at 4°C overnight, washed four times (10 minutes each) in PBS-Tween, incubated with horseradish peroxidase-labeled goat anti-rabbit IgG (1:2,000; Sigma) at room temperature for 120 minutes, and washed four times (10 minutes each) in PBS-Tween.

Techniques: Expressing