Journal: Life Science Alliance

Article Title: Paneth cell α-defensin misfolding correlates with dysbiosis and ileitis in Crohn’s disease model mice

doi: 10.26508/lsa.201900592

Figure Lengend Snippet: (A, B) Representative transmission electron microscopy images of Paneth cells at the base of ileal crypts in (A) ICR and (B) SAMP1/YitFc mice. Scale bars indicate 2 μm. (C, D) Quantitative analysis of (C) granule number and (D) ER lumen diameter in Paneth cells (n = 3/each week for SAMP1/YitFc mice). For the measurements, three Paneth cells were randomly selected from each mouse. (E) SDS–PAGE Western blot analysis of ER stress markers, pIRE1α, ATF4, cleaved-ATF6, and GRP78 in ileal crypts (n = 4/each group). Total-IRE1α and HPRT1 was used as loading control. (F) Relative expression level of ER stress markers calculated from the band intensity. Error bars represent mean ± SEM. (C, D, F) Statistical significance was evaluated by t test in (C, D), and one-way ANOVA followed by Tukey’s post hoc test in (F). P < 0.05 was considered statistically significant. * P < 0.05, † P < 0.01, § P < 0.001. E, ER; G, granules; N, nucleus; n.s., not significant.

Article Snippet: After transfer, the membranes were blocked by 5% BSA (Sigma-Aldrich) in 0.1% TBS-T at 4°C for overnight and then reacted with the following primary antibodies: anti-ATF4 (1/1,000, #11815; Cell Signaling Technology), anti-phospho-IRE1α (1 μg/ml, NB100-2323; Novus Biologicals), anti-total IRE1α (1 μg/ml, NB100-2324; Novus Biologicals), anti-ATF6 (1 μg/ml, NBP1-40256; Novus Biologicals), anti-GRP78 (1/1,000, #3177; Cell Signaling Technology), and anti-HPRT1 (0.018 μg/ml, ab109021; Abcam) at 4°C overnight.

Techniques: Transmission Assay, Electron Microscopy, SDS Page, Western Blot, Control, Expressing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Sarco(endo)plasmic reticulum Ca 2+ -ATPase 2b is a major regulator of endoplasmic reticulum stress and glucose homeostasis in obesity

doi: 10.1073/pnas.1012044107

Figure Lengend Snippet: SERCA2b protein and mRNA levels are decreased in the liver of obese mice. (A) SERCA2b and tubulin immunoblotting from the whole liver lysates of lean WT and ob/ob mice. (B) Quantification of the Western blot showing the ratio of SERCA2b to tubulin. (C) SERCA2b and IRE1α immunoblotting in the isolated liver ER fractions of WT and ob/ob mice. (D) Quantification of the blot in the ratio of SERCA2b to IRE1α. (E) mRNA levels of SERCA2b in the liver of WT and ob/ob mice. 18S was used as a control. (F) Western blot for SERCA2b and tubulin in the whole liver lysates of mice that were kept on normal or high fat diet for 16 wk. (G) Quantification of the blot showing the ratio of SERCA2b/tubulin. (H) Quantitative PCR for SERCA2b in the liver of normal diet and high-fat diet mice. (I) Fold increase of SERCA2b mRNA expression during 1, 3, and 5 h of refeeding after 24 h of fasting in lean WT mice. (J) Fold increase of SERCA2b mRNA expression during refeedings after 24 h of fasting in ob/ob mice. (K–M) Seven-week-old male ob/ob mice were injected with Ad-LacZ or Ad-XBP1s (4 × 108 pfu/g) through a tail vein. (K) Western blots for the levels of XBP1s and Akt as a control in the liver of adenovirus-injected ob/ob mice on day 7 after injection. (L) SERCA2b and tubulin levels in the liver of Ad-LacZ or Ad-XBP1s injected mice on day 7 after injection. (M) mRNA levels of SERCA2b in the liver of Ad-LacZ and Ad-XBP1s injected mice with 18S level as a control. Error bars are SEMs; P values were determined by Student t test (*P < 0.05, **P < 0.01, ***P < 0.001).

Article Snippet: Antibodies for phospho-IRE1α Ser724 and total IRE1α were purchased from Novus Biologicals.

Techniques: Western Blot, Isolation, Control, Real-time Polymerase Chain Reaction, Expressing, Injection

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Sarco(endo)plasmic reticulum Ca 2+ -ATPase 2b is a major regulator of endoplasmic reticulum stress and glucose homeostasis in obesity

doi: 10.1073/pnas.1012044107

Figure Lengend Snippet: Overexpression of SERCA2b in the liver of ob/ob mice increases glucose tolerance and establishes euglycemia. (A–J) Eight-week-old male ob/ob mice were injected with Ad-LacZ or Ad-SERCA2b (5.8 × 107 pfu/g) through a tail vein. (A) SERCA2b immunoblotting in the liver of Ad-LacZ or Ad-SERCA2b-injected ob/ob mice. (B) mRNA levels of SERCA2b in the liver of ob/ob mice that were injected with Ad-LacZ or Ad-SERCA2b. (C) Blood glucose level (mg/dL) on day 3 after injection at 6 h of fasting. (D) Serum insulin level (ng/mL) on postinjection day 8. (E) GTT was performed with i.p. injection of 0.5 g/kg of glucose on day 4 after injections. (F) ITT was performed with i.p. injection of insulin (2 IU/kg) on postinjection day 7 after 6 h fasting. (G) Body weight (in g) at postinjection days 3 and 5. (H) Phospho-IRE1αSer724 and total IRE1 protein levels in the liver of Ad-LacZ and Ad-SERCA2b-injected ob/ob mice. (I) Quantification of phospho/total IRE1 ratio. (J) qPCR analysis of the mRNA levels of PDI, ERDJ4, calnexin, calreticulin, EDEM, ERO1α, HERP, GRP58, and GRP78. (K) Gene expression level of G6Pase, PEPCK, and PGC1α was analyzed by qPCR in the liver of Ad-LacZ or Ad-SERCA2b-injected ob/ob mice. Error bars are means SEM; P values were determined by Student t test (*P < 0.05, **P < 0.01, ***P < 0.001).

Article Snippet: Antibodies for phospho-IRE1α Ser724 and total IRE1α were purchased from Novus Biologicals.

Techniques: Over Expression, Injection, Western Blot, Gene Expression