mouse anti hspb1  (Proteintech)

Journal: Translational Vision Science & Technology

Article Title: AAV2-Mediated Expression of HspB1 in RGCs Prevents Somal Damage and Axonal Transport Deficits in a Mouse Model of Ocular Hypertension

doi: 10.1167/tvst.11.11.8

Figure Lengend Snippet: Prophylactic effects of AAV2- HSPB1 in a mouse model of glaucoma. (A) The AAV2- HSPB1 injection timeline for a mouse model of glaucoma is shown. MBs were injected into the anterior chamber of one eye two weeks after the intravitreal injection of AAV2- HSPB1 (1 µL from the stock of 1 × 10 12 viral genomes/mL and 2 µL from the stock of 2.5 × 10 12 viral genomes/mL). Four weeks later, retinal flatmounts were fixed and immunostained for RGCs. (B) The IOP of MB-injected eyes was significantly elevated one week post-MB injection (WPI). (C) After immunostaining whole retinal flatmounts with Brn3a, four areas in the mid-periphery were selected to determine RGC density. Confocal microscopic images were captured from the selected retinal areas in the contralateral (control), vehicle-treated, AAV2-control-treated, and AAV2- HSPB1 -treated groups at four WPI. Scale bar: 100 µm. (D) The bar graph shows the number of RGCs. (E) Mice were injected as above, and after four weeks, Alexa Fluor 555-conjugated CT-B (1 µL, 0.2%) was injected intravitreally. The animals were killed 24 hours after CT-B injection and the optic nerves were dissected out. Confocal microscopic (TRITC filter) images were captured along the length of the optic nerve. Scale bar: 1000 µm. (F) Quantification of the CT-B intensity. Data represent the mean ± SEM of four to eight independent experiments. Each data point represents one animal. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. ns, not significant.

Article Snippet: The primary antibodies were as follows: HspB1 (Cat no. 2402S; Cell Signaling Technology, Danvers, MA, USA), HspB4 (Cat no. ADI-SPA-221; Enzo Life Sciences, Farmingdale, NY, USA), HspB5 (Cat no. ABN185; Millipore, Billerica, MA, USA), and HspB6 (Cat no. ADI-SPA-796, Enzo Life Sciences).

Techniques: Injection, Immunostaining, Control

Journal: Translational Vision Science & Technology

Article Title: AAV2-Mediated Expression of HspB1 in RGCs Prevents Somal Damage and Axonal Transport Deficits in a Mouse Model of Ocular Hypertension

doi: 10.1167/tvst.11.11.8

Figure Lengend Snippet: Therapeutic effects of AAV2- HSPB1 in a mouse model of glaucoma: Effect on the time-dependent progression of RGC death. (A) The AAV2- HSPB1 injection timeline for a mouse model of glaucoma is shown. MB were injected into the anterior chamber of one eye one week before the AAV2- HSPB1 injection (1 µL from the stock of 1 × 10 12 viral genomes/mL). Five weeks later, retinal flat mounts were fixed and immunostained for RGCs and HspB1. (B) The IOP of MB-injected eyes was significantly elevated at one WPI and remained significantly higher for six weeks. (C) Retinal flat mounts were immunostained for HspB1 and RPBMS (for RGCs). AAV2- HSPB1 injection resulted in robust expression of HspB1 in RGCs at 5 WPI. (D) The Brn3a +ve RGCs from the mid-peripheral retina are shown for each group. Confocal microscopic images were captured in contralateral (control), MB + vehicle-treated, MB + AAV2-control-treated, and MB + AAV2- HSPB1 -treated groups at six WPI. (E) The bar graph shows the quantification of Brn3a +ve RGC loss in retinal flat mounts. Data represent the mean ± SEM of 5-7 independent experiments. Each data point represents one animal. (F) Effect of AAV2- HSPB1 treatment on the time-dependent RGC loss in the mid-peripheral retina one, two, four, and six WPI is shown. * P < 0.05; ** P < 0.01; *** p < 0.001; **** P < 0.0001. ns, not significant. Scale bar: 100 µm.

Article Snippet: The primary antibodies were as follows: HspB1 (Cat no. 2402S; Cell Signaling Technology, Danvers, MA, USA), HspB4 (Cat no. ADI-SPA-221; Enzo Life Sciences, Farmingdale, NY, USA), HspB5 (Cat no. ABN185; Millipore, Billerica, MA, USA), and HspB6 (Cat no. ADI-SPA-796, Enzo Life Sciences).

Techniques: Injection, Expressing, Control

Journal: Translational Vision Science & Technology

Article Title: AAV2-Mediated Expression of HspB1 in RGCs Prevents Somal Damage and Axonal Transport Deficits in a Mouse Model of Ocular Hypertension

doi: 10.1167/tvst.11.11.8

Figure Lengend Snippet: Therapeutic effects of AAV2- HSPB1 in a mouse model of glaucoma: Effects on axonal transport and glial activation. MB were injected into the anterior chamber of the eye one week before and two weeks after AAV2-control or AAV2- HSPB1 injection. (A) Six weeks after MB injection, mice were intravitreally injected with Alexa Fluor 555-conjugated CT-B (1 µL, 0.2%), and the optic nerve was isolated. Confocal microscopic images were captured along the entire length of the optic nerve. Scale bar: 1000 µm. (B) Quantification of the CT-B intensity. (C, D) Inhibitory effect of AAV2- HSPB1 on glial activation. Six weeks after MB injection, representative images of the retinal GCL layer showing (C) Iba1 (activated microglial marker, purple) and (D) GFAP (activated astrocyte marker, green) in retinal flat mounts. Data represent the mean ± SEM of six to nine (A, B) and three to four (C, D) independent experiments. Each data point represents one animal. * P < 0.05; ** P < 0.01. ns, not significant. Scale bar: 100 µm.

Article Snippet: The primary antibodies were as follows: HspB1 (Cat no. 2402S; Cell Signaling Technology, Danvers, MA, USA), HspB4 (Cat no. ADI-SPA-221; Enzo Life Sciences, Farmingdale, NY, USA), HspB5 (Cat no. ABN185; Millipore, Billerica, MA, USA), and HspB6 (Cat no. ADI-SPA-796, Enzo Life Sciences).

Techniques: Activation Assay, Injection, Control, Isolation, Marker

Journal: Translational Vision Science & Technology

Article Title: AAV2-Mediated Expression of HspB1 in RGCs Prevents Somal Damage and Axonal Transport Deficits in a Mouse Model of Ocular Hypertension

doi: 10.1167/tvst.11.11.8

Figure Lengend Snippet: Long-term efficacy of AAV2- HSPB1 in a mouse model of glaucoma: Effects on axonal transport and glial activation. (A) The AAV2- HSPB1 injection timeline for a mouse model of glaucoma is shown. MB were injected into the anterior chamber of the eye at the start of the experiment and at three weeks and six weeks. AAV2-control or AAV2- HSPB1 (1 µL from the stock of 1 × 10 12 viral genomes/mL) was injected intravitreally one WPI. (B) The IOP of MB-injected eyes was significantly elevated one WPI, remained significantly higher over ten weeks and then gradually decreased between 10-20 weeks. (C) After 20 weeks, retinas were isolated, retinal flat mounts were fixed and immunostained for RGCs (Brn3a, green). Confocal microscopic images were captured from the mid-peripheral regions of the retina in the contralateral (control), MB + AAV2-control-treated, and MB + AAV2- HSPB1 -treated groups at 20 weeks. (D) Bar graph showing Brn3a +ve RGCs in retinal flat mounts. (E) Representative pERG response at 20 weeks. (F) Quantification of the P1 amplitude of pERG. Data represent the mean ± SEM of five to 11 independent experiments. Each data point represents one animal. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns = not significant. Scale bar: 50 µm.

Article Snippet: The primary antibodies were as follows: HspB1 (Cat no. 2402S; Cell Signaling Technology, Danvers, MA, USA), HspB4 (Cat no. ADI-SPA-221; Enzo Life Sciences, Farmingdale, NY, USA), HspB5 (Cat no. ABN185; Millipore, Billerica, MA, USA), and HspB6 (Cat no. ADI-SPA-796, Enzo Life Sciences).

Techniques: Activation Assay, Injection, Control, Isolation

Journal: Cells

Article Title: The Effect of Heat Shock on Myogenic Differentiation of Human Skeletal-Muscle-Derived Mesenchymal Stem/Stromal Cells.

doi: 10.3390/cells11203209

Figure Lengend Snippet: Figure 6. The effect of heat stimulus on the CD56(−) and CD56(+) cells. The level of HSF1 in CD56(−) (A) and CD56(+) (B) cells estimated by flow cytometer. (C). The levels of HSP27, HSP70/72, HSP90, desmin and vimentin in CD56(−) and CD56(+) cells after the 42 ◦C heat for 1 h and different recover periods (0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, 24 h) under the normal growth conditions. Data are shown as mean ± standard deviation (SD) from not less than three repeats (n = 3) and three cell cultures calculated using the Excel software and are significant at * p ≤0.05. Data show representative WB micrographs of both cell types. The protein level has been equalized using a Lowry protein kit. Control cells were not heat stimulated.

Article Snippet: Later, the membrane was washed with TBST (Tris buffered saline with 20% Tween 20) and blocked at RT with 3% BSA in TBST for 1 h. Antibodies against: HSP70/72 (genes HSPA1A/HSPA1B) (ADI-SPA-810-F) (Enzo, New York, NY, USA), HSP27 (G31) #2402 (gene HSPB1) (Cell Signaling); HSP90 (C45G5) #4877 (gene HSP90AA1) (Cell Signaling), desmin (5332T, Cell Signaling Technology, Danvers, MA, USA), vimentin (Sc-7557, Santa Cruz Biotechnology, Dallas, TX, USA), HSF1 (12972S, Cell Signaling Technology, Danvers, MA, USA), beta-actin (PA1-46296, Thermo Fisher Scientific, Waltham, MA, USA) were used to identify protein levels.

Techniques: Cytometry, Standard Deviation, Software, Control