Journal: Neural Regeneration Research

Article Title: PTEN inhibitor bisperoxovanadium protects against noise-induced hearing loss

doi: 10.4103/1673-5374.358606

Figure Lengend Snippet: bpV prevents noise-induced reductions in PI3K-Akt signaling in OHCs. (A, B) Representative images showing the reduced levels of PI3K-p85α and -p110α (red, Alexa Fluor 594) in OHCs after noise exposure, as detected by immunofluorescence staining. Pretreatment with bpV protected against these effects. Scale bars: 20 μm. (C, D) Quantification of PI3K-p85α and -p110α levels in OHCs in noise (PTS) and noise + bpV (PTS + bpV) groups. (E, F) Representative images and quantification analyses showed that p-Akt levels were reduced in OHCs after noise exposure. (G) mRNA expression levels of PI3K and Akt were detected by real-time PCR in control, noise, and noise + bpV groups. (H) Protein expression of PI3K-p85α, -p110α, p-Akt and Akt were determined by western blotting. (I–K) Quantification of PI3K-p85α and -p110α relative to GAPDH and of p-Akt/Akt. Expression of p110α decreased after noise exposure compared with that in the control group. There was no change in total Akt level between the three groups. Data are presented as the mean ± SD. The experiment was repeated three times. * P < 0.05, ** P < 0.01, *** P < 0.001 (unpaired Student’s t -test). bpV: Bisperoxovanadium; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; IHC: inner hair cell; OHC: outer hair cell; p-Akt: phospho-Akt; PCR: polymerase chain reaction; PI3K: phosphoinositide-3 kinase; PTS: permanent threshold shift.

Article Snippet: The primary antibodies were monoclonal rabbit anti-PTEN (Cell Signaling Technology, Cat# 9188, RRID: AB_2253290), monoclonal rabbit anti-PI3K-p110α (Cell Signaling Technology, Cat# 4249, RRID: AB_2165248), polyclonal rabbit anti-PI3K-p85α (Cell Signaling Technology, Cat# 4292, RRID: AB_329869), monoclonal rabbit anti-phospho-Akt (Ser 473) (Cell Signaling Technology, Cat# 4060, RRID: AB_2315049) and polyclonal rabbit anti-4 hydroxynonenal (4HNE; Abcam, Cambridge, UK, Cat# ab46545, RRID: AB_722490).

Techniques: Immunofluorescence, Staining, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Polymerase Chain Reaction

Journal: Neural Regeneration Research

Article Title: Argon preconditioning protects neuronal cells with a Toll-like receptor-mediated effect

doi: 10.4103/1673-5374.355978

Figure Lengend Snippet: Argon preconditioning reduces rotenone-induced protein expression of Bax and increases Bcl-2, but the effect is abolished by OxPAPC. (A, C) Densitometric analysis of western blots for Bax (A) and Bcl-2 (C) protein expression. Between-group comparisons were performed with a one-way analysis of variance using the post hoc Holm Sidak test ( n = 6; mean ± SD; * P < 0.05, ** P < 0.01, *** P < 0.001). (B, D) Representative western blot images of six independent experiments that showed similar results demonstrating the influence of rotenone, argon preconditioning, and inhibition with OxPAPC on Bax (B) and Bcl-2 (D).

Article Snippet: After protein transfer to polyvinylidene difluoride membranes (Immobilon-P; Millipore, Schwalbach, Germany), the membranes were blocked with 5% skim milk in Tween20/PBS (TBST) or BSA (bovine serum albumin) and incubated in the recommended dilution of protein-specific antibody (p44/42 MAP kinase (phospho-ERK1/2 [extracellular-signal regulated kinase]) (Thr202/Tyr204), rabbit, 1:2000, Cell Signaling Technology, Danvers, MA, USA, Cat# 9101, RRID: AB_331646; phospho-NF-κB (Ser536) (93H1), rabbit, 1:1000, Cell Signaling Technology, Cat# 3033, RRID: AB_331284; phospho-Akt (protein kinase B) antibody (Ser473), rabbit, 1:1000, Cell Signaling Technology, Cat# 9271, RRID: AB_329825; Bax antibody, rabbit, 1:1000, Cell Signaling Technology, Cat# 2772, RRID: AB_10695870; Bcl-2 antibody, rabbit, 1:1000, Cell Signaling Technology, Cat# 2876, RRID: AB_2064177; and cleaved caspase-3 antibody (Asp175), rabbit, 1:500, Cell Signaling Technology, Cat# 9661, RRID: AB_2341188; NF-E2-related factor 2 (Nrf2) antibody [nuclear factor erythroid 2-related factor 2, phosphor-S40], rabbit, 1:60000, Abcam, Cat# ab76026, RRID: AB_1524049).

Techniques: Expressing, Western Blot, Inhibition

Journal: Life Science Alliance

Article Title: MAP4K3 inhibits Sirtuin-1 to repress the LKB1–AMPK pathway to promote amino acid-dependent activation of the mTORC1 complex

doi: 10.26508/lsa.202201525

Figure Lengend Snippet: List of antibodies.

Article Snippet: Phospho-Akt (Ser473) , 9271 , Cell Signaling.

Techniques:

Journal: Scientific Reports

Article Title: Skeletal muscle overexpression of sAnk1.5 in transgenic mice does not predispose to type 2 diabetes

doi: 10.1038/s41598-023-35393-0

Figure Lengend Snippet: Intra-peritoneal glucose (IPGTT), insulin levels and intra-peritoneal insulin tolerance tests (IPITT) in chow-fed mice. ( a ) IPGTT was performed on WT and transgenic mice at 2, 7, 10 and 12 months of age (n = 25 WT and 25 Tg sAnk1.5/+ ; 22 WT and 24 Tg sAnk1.5/+ ; 25 WT and 25 Tg sAnk1.5/+ ; 15 WT and 16 Tg sAnk1.5/+ , respectively). Mice were fasted overnight for 17 h, and glucose (2 g/kg) was administered through intraperitoneal injection. Blood glucose concentration was measured at 0, 30-, 60-, 120-, and 180-min following injection. ( b ) AUC ± SD calculated from glycemic curves reported in ( a ). ( c ) Serum insulin levels (ng/ml ± SD) in WT and Tg sAnk1.5/+ mice following 17 h of fasting, and 15 and 60 min after glucose administration (2 g/kg). ( d ) IPITT was performed on 12-month-old WT and transgenic mice. Mice were fasted daily for 5 h, and insulin (1 U/kg) was administered through intraperitoneal injection. Blood glucose concentration was measured at 0, 15-, 30-, 60-, 90-, and 120 min following injection. e : AUC ± SD calculated from glycemic curves reported in ( d ). ( f ) Western blot analyses of AKT phosphorylation at serine 473 in the gastrocnemius muscle from WT and Tg sAnk1.5/+ mice. ( g ) Densitometric analysis of p-AKT Ser473 , p-AKT Thr308 (see also Supplementary Fig. online) and total AKT, using actin (ponceau S staining) as a normalizer, is expressed as fold levels of pAKT/totalAKT ratio ± SD, relative to WT mice. *p < 0.01, Student’s t-test. Original blots/gels are reported in Supplementary Fig. online.

Article Snippet: The following primary antibodies were used: polyclonal rabbit anti-SERCA TRY2, home-made, used at 1:2000 ; polyclonal rabbit anti-sAnk1.5, home-made, used at 1:1000 ; monoclonal anti-calsequestrin-1, clone VIIID12, cat. number MA3-913, Thermo Fisher Scientific (Waltham, MA), used at a 1:1000; polyclonal rabbit anti-AKT(pan), cat. n. 4691, polyclonal rabbit anti-phospho-AKT (Ser473), cat. n. 4060, and polyclonal rabbit anti-phospho-AKT (Thr308), cat. n. 2965, were all from Cell Signaling Technology (Danvers, MA) and used at 1:1000; rabbit anti-Sln, 18395-1-AP was from Proteintech (Rosemont, IL) and used at 1:1000; mouse anti-Pln, MA3-922 was from Thermo Fisher Scientific and used 1:1000.

Techniques: Transgenic Assay, Injection, Concentration Assay, Western Blot, Staining

Journal: Scientific Reports

Article Title: Skeletal muscle overexpression of sAnk1.5 in transgenic mice does not predispose to type 2 diabetes

doi: 10.1038/s41598-023-35393-0

Figure Lengend Snippet: IPGTT and IPITT on high fat fed mice. 2-month-old mice were divided into four groups and fed either chow (standard diet; SD) or a high-fat diet (HFD) for twelve weeks (n = 18 SD WT, 18 SD Tg sAnk1.5/+ , 18 HFD WT, 20 HFD Tg sAnk1.5/+ ). ( a ) IPGTT of 2-month-old WT and Tg sAnk1.5/+ mice before starting a high-fat diet (n = 36 WT and 38 Tg sAnk1.5/+ ). ( b ) IPGTT of WT and Tg sAnk1.5/+ mice at the end of the high-fat diet period. ( c ) IPITT of WT and Tg sAnk1.5/+ mice at the end of the high-fat diet period. ( d ) areas under the curves (AUC ± SD) calculated from glycemic curves at 2 months of age reported in ( a ). € AUC ± SD calculated from glycemic curves of chow (SD)- and high fat (HFD) fed mice at the end of the high-fat diet period reported in ( b ). ( f ) AUC ± SD calculated from the IPITT experiment reported in ( c ). * and # refer to WT SD vs WT HFD and Tg sAnk1.5/+ SD vs Tg sAnk1.5/+ HFD, respectively; * and # , ** and ## , ***, **** and #### p < 0.05, < 0.01, < 0.001, < 0.0001, respectively; n.s. = not significant. ( g ) representative western blot analysis of AKT phosphorylation at serine 473 in the gastrocnemius of chow-fed (SD) and high-fat diet-fed (HFD) WT and Tg sAnk1.5/+ mice. ( h ) Densitometric analysis of p-AKT Ser473 and total AKT, using actin (ponceau S staining) as a normalizer, for both dietetic regimens. Data are expressed as pAKT 473 /total AKT ratio ± SD relative to WT diet-matched mice. Densitometric analysis of sAnk1.5 signal intensity relative to WT diet-matched mice is also reported. *p < 0.05, Student’s t-test. Original blots/gel are reported in Supplementary Fig. online.

Article Snippet: The following primary antibodies were used: polyclonal rabbit anti-SERCA TRY2, home-made, used at 1:2000 ; polyclonal rabbit anti-sAnk1.5, home-made, used at 1:1000 ; monoclonal anti-calsequestrin-1, clone VIIID12, cat. number MA3-913, Thermo Fisher Scientific (Waltham, MA), used at a 1:1000; polyclonal rabbit anti-AKT(pan), cat. n. 4691, polyclonal rabbit anti-phospho-AKT (Ser473), cat. n. 4060, and polyclonal rabbit anti-phospho-AKT (Thr308), cat. n. 2965, were all from Cell Signaling Technology (Danvers, MA) and used at 1:1000; rabbit anti-Sln, 18395-1-AP was from Proteintech (Rosemont, IL) and used at 1:1000; mouse anti-Pln, MA3-922 was from Thermo Fisher Scientific and used 1:1000.

Techniques: Western Blot, Staining