Journal: The Journal of biological chemistry

Article Title: Arrest of mycobacterial phagosome maturation is caused by a block in vesicle fusion between stages controlled by rab5 and rab7.

doi: 10.1074/jbc.272.20.13326

Figure Lengend Snippet: FIG. 6. GTP binding and protein immunoblot analysis of LBC and MPC indicating the presence of rab5 and the absence of rab7 in MPC samples. Equivalent amounts of phagosomes (with the exception of slightly overloaded LBC sample at 168 h) were separated by SDS-PAGE, electroblotted to Immobilon P membranes, and probed successively with [a-32P]GTP (panel A), affinity-purified antibody to rab5a (panel B), affinity-purified antibody against rab7 (panel C), and rat monoclonal antibody to Lamp2 (panel D). Note that the top band of the apparent triplet on the GTP overlay (panel A) corresponds to the location of the polypeptide recognized by the antibody to rab5a.

Article Snippet: For immunoblots, membranes were incubated with affinity-purified antibodies for rab4, rab5a, and rab7 prepared and characterized as described by Zerial et al. (40), or rat monoclonal antibodies to murine Lamp1 (clone 1D4B), Lamp2 (clone ABL-93) (Developmental Hybridoma Bank, NICHHD, Iowa City, IA) transferrin receptor (Biosource International), or rabbit anti-actin (Sigma).

Techniques: Binding Assay, Western Blot, SDS Page, Affinity Purification

Journal: Cell reports

Article Title: Thbs1 regulates skeletal muscle mass in a TGFβ-Smad2/3-ATF4-dependent manner

doi: 10.1016/j.celrep.2024.114149

Figure Lengend Snippet: (A) Representative western blots for Thbs1, Thbs4, ATF6α-N (50 kDa, nuclear), BiP, calreticulin (Calret.), PERK, ATF4, IRE1α, fibroblast growth factor 21 (FGF21), LC3b, p62, LAMP1, LAMP2, and LIMPII from quadriceps protein extracts from Ntg and skeletal muscle-specific Thbs1 Tg, Thbs4 Tg, and ATF6α Tg mice at 6 weeks of age. Gapdh serves as a processing and loading control; n = 3 biologically independent animals per genotype. Red boxed areas show specific expression changes in Thbs1 Tg muscle. (B and C) Representative western blots for LC3b, p62, and Gapdh as a loading control in 6-week-old quadriceps protein extracts from Ntg and Thbs1 Tg mice treated with water as vehicle (Veh.) or 0.4 mg/kg/day colchicine (Colch.) for 2 days to impact autolysosome content/activity. (C) Quantitative analysis of LC3b-II protein levels relative to Gapdh from the experiment shown in (B). Error bars denote ±SEM from n = 3 biologically independent animals per genotype and per treatment. * p < 0.05, ** p < 0.01, *** p < 0.001 by one-way ANOVA and Tukey’s multiple comparison test. (D) 20S chymotrypsin-like proteasome activity (pmol/[min·μg]) in quadriceps tissue of Ntg and skeletal muscle-specific Thbs1 Tg, Thbs4 Tg, and ATF6α Tg mice at 6 weeks of age. Error bars denote ±SEM from n = 4–7 biologically independent animals per indicated genotype. * p < 0.05, ** p < 0.001 by one-way ANOVA and Tukey’s multiple comparison test. (E) Representative western blot for ubiquitin-conjugated proteins (Ubiq.) in quadriceps tissue of the indicated genotypes of mice at 6 weeks of age. Gapdh serves as loading control; n = 3 biologically independent animals per genotype. Quantitative analysis of this experiment is presented in . (F) Representative western blots for Thbs1, Thbs2, ATF6α-N (50 kDa, nuclear), PERK, ATF4, and LAMP2 from quadriceps protein extracts from Ntg and skeletal muscle-specific Thbs1 Tg and Thbs2 Tg mice at 12 weeks of age. Gapdh serves as loading control; n = 3 biologically independent animals per genotype. (G) Immunohistochemistry micrographs showing wheat germ agglutinin (WGA, green) and DAPI (blue) in quadriceps of Ntg, Thbs1 Tg, and Thbs2 Tg at 12 weeks of age. Scale bars represent 100 μm. (H) Myofiber cross-sectional area (CSA) in Ntg, Thbs1 Tg, and Thbs2 Tg quadriceps at 12 weeks of age as shown in (G). Error bars denote ±SEM from n = 4–5 biologically independent animals per genotype. *** p < 0.001 by one-way ANOVA and Tukey’s multiple comparison test. See also and .

Article Snippet: Primary antibodies used in this study were: Akt (Cell Signaling Technology, Cat# 9272 at 1:1000), p-Akt Ser473 (Cell Signaling Technology, Cat# 4060 at 1:1000), AMPKα (Cell Signaling Technology, Cat# 2532 at 1:1000), p-AMPKα Thr172 (Cell Signaling Technology, Cat# 2535 at 1:1000), ATF4 (Cell Signaling Technology, Cat# 11815 and LSBio LifeSpan Biosciences, Cat# LS-B6361; both at 1:1000), ATF6α (SAB SignalWayAntibody LLC, Cat# 24383; at 1:1000), BiP (Sigma, Cat# GG8918; at 1:1000), calreticulin (Cell Signaling Technology, Cat# 2891; at 1:1000), β-dystroglycan (developmental Studies Hybridoma Bank, cat# MANDAG2 clone 7D11; 1:100), p-4E-BP1 Thr37/46 (Cell Signaling Technology, Cat# 2855 at 1:1000), 4E-BP1 (Cell Signaling Technology, Cat# 9644 at 1:1000), FGF21 (R&D Systems, Cat# AF3057; at 1:500), Foxo1 (Cell Signaling Technology, Cat# 2880 at 1:1000), p-Foxo1 ser256 (Cell Signaling Technology, Cat# 9461 at 1:1000), Foxo3 (Cell Signaling Technology, Cat# 2497 at 1:1000), p-Foxo3 ser413 (Cell Signaling Technology, Cat# 8174 at 1:1000), p-Foxo3 ser 253 (Cell Signaling Technology, Cat# 9466 at 1:1000), IRE1α (Cell Signaling Technology, Cat# 3294; at 1:1000), β1D-integrin (Millipore, MAB1900; at 1:500), LAMP1 (Development Studies Hybridoma Bank, Cat# 1D4B; at 1:100), LAMP2 (Development Studies Hybridoma Bank, Cat# ABL-93; at 1:100), LC3b (Cell Signaling Technology, Cat# 3868; at 1:1000), mTOR (Cell Signaling Technology, Cat# 2972 at 1:1000), p -mTOR ser2448 (Cell Signaling Technology, Cat# 2971 at 1:1000), p62 (Sigma, Cat# p0067 at 1:1000), LIMPII (Abcam, Cat# ab176317; at 1:1000), p-p70 S6K Thr389 (Cell Signaling Technology, Cat# 9205 at 1:1000), p70 S6K (Cell Signaling Technology, Cat# 2708 at 1:1000),PERK (Cell Signaling Technology, Cat# 3192; at 1:1000), anti-puromycin (Sigma, Cat# MABE343; at 1:1000); α-sarcoglycan (Development Studies Hybridoma Bank, Cat# IVD3(1)A9; at 1:100), β-sarcoglycan (Novus Biologicals, Cat# NBP1–90300; at 1:1000); δ-sarcoglycan (Abcam, Cat# ab137101; at 1:500); Smad2 (Cell Signaling Technology, Cat# 5339; at 1:1000); Smad3 (Abcam, Cat# ab40854; at 1:500); P-Smad2 Ser465/467/P-Smad3 Ser423/425 (Signaling Technology, Cat# 8828; at 1:1000); p-Smad3 Ser423/425 (Signaling Technology, Cat# 9520; at 1:1000); TGFβ RII (Santa Cruz Biotechnology, Cat# sc-17799; at 1:200); Thbs1 (R&D Systems, Cat# AF3074; at 1:500 and ThermoFisher Scientific, Cat# MA5–13395; at 1:1000); Thbs2 (R&D Systems, Cat# MAB1635; at 1:500); Thbs4 (R&D Systems, Cat# AF2390; at 1:500); Ubiquitin (Santa Cruz Biotechnology, Cat# sc-8017; at 1:200); and glyceraldehyde 3-phosphate dehydrogenase (Gapdh; Fitzgerald, Cat# 10R-G109A; at 1:10000) which served as overall loading control.

Techniques: Western Blot, Control, Expressing, Activity Assay, Comparison, Ubiquitin Proteomics, Immunohistochemistry

Journal: Cell reports

Article Title: Thbs1 regulates skeletal muscle mass in a TGFβ-Smad2/3-ATF4-dependent manner

doi: 10.1016/j.celrep.2024.114149

Figure Lengend Snippet: (A) Representative western blot analysis of Thbs1, Smad3, Smad2, ATF4, FGF21, LC3b, p62, LAMP2, and LIMPII in 12-week-old quadriceps protein extracts from Smad2/3 fl/fl , Smad2/3 fl/fl - Myl1 -Cre ( Smad2/3 mKO ), Thbs1 Tg Smad2/3 fl/fl , and Thbs1 Tg Smad2/3 mKO mice. Gapdh serves as loading control; n = 2 biologically independent animals per genotype. Red arrowhead indicates ATF4. (B and C) 20S chymotrypsin-like proteasome activity (pmol/[min·μg]; B) and quadriceps (Quad) weight normalized to tibia length (TL; C) from the indicated genotypes of mice at 12 weeks of age based on the color-coded legend shown on the right. Error bars denote ±SEM. The number of biologically independent animals per genotype are indicated in the graphs. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 by one-way ANOVA and Tukey’s multiple comparison test. (D) Western blots for phospho-Smad3 (Ser423/425), total Smad3, and ATF4 in gastrocnemius from 16-day-old rat pups injected with Adβgal or AdSmad3 on postnatal day 2, followed by a second “boost” injection 72 h later (3E8 viral particles per injection). Gapdh serves as loading control; n = 3 biologically independent animals per genotype. (E and F) Gastrocnemius (Gastroc) weight normalized to tibia length (TL) from 16-day-old rat pups injected with Adβgal or AdSmad3 (E) or injected with Adβgal, AdThbs1, AdSmad6, and/or AdSmad7 (F), following the timeline described for (D) (all 3E8 viral particles per injection). Error bars denote ±SEM from n = 5–6 biologically independent animals per treatment group. * p < 0.05 by two-tailed unpaired Student’s t test for (E); * p < 0.05, ** p < 0.01, *** p < 0.001 by one-way ANOVA and Tukey’s multiple comparison test for (F). (G) Western blot analysis for Thbs1, ATF4, FGF21, LC3b, p62, LAMP2, and Gapdh in 12-week-old quadriceps protein extracts from Atf4 fl/fl , Atf4 fl/fl - Myl1 -Cre ( Atf4 mKO ), Thbs1 Tg Atf4 fl/fl , and Thbs1 Tg Atf4 mKO mice; n = 2 biologically independent animals per genotype. (H and I) 20S chymotrypsin-like proteasome activity (pmol/[min·μg]; H) and quadriceps (Quad) weight normalized to tibia length (TL; I) from the indicated genotypes at 12 weeks of age. Error bars denote ±SEM from n = 3 biologically independent animals per genotype in (H) and n = 6–8 biologically independent animals per genotype for (I). * p < 0.05, ** p < 0.01, *** p < 0.001, by one-way ANOVA and Tukey’s multiple comparison test. See also .

Article Snippet: Primary antibodies used in this study were: Akt (Cell Signaling Technology, Cat# 9272 at 1:1000), p-Akt Ser473 (Cell Signaling Technology, Cat# 4060 at 1:1000), AMPKα (Cell Signaling Technology, Cat# 2532 at 1:1000), p-AMPKα Thr172 (Cell Signaling Technology, Cat# 2535 at 1:1000), ATF4 (Cell Signaling Technology, Cat# 11815 and LSBio LifeSpan Biosciences, Cat# LS-B6361; both at 1:1000), ATF6α (SAB SignalWayAntibody LLC, Cat# 24383; at 1:1000), BiP (Sigma, Cat# GG8918; at 1:1000), calreticulin (Cell Signaling Technology, Cat# 2891; at 1:1000), β-dystroglycan (developmental Studies Hybridoma Bank, cat# MANDAG2 clone 7D11; 1:100), p-4E-BP1 Thr37/46 (Cell Signaling Technology, Cat# 2855 at 1:1000), 4E-BP1 (Cell Signaling Technology, Cat# 9644 at 1:1000), FGF21 (R&D Systems, Cat# AF3057; at 1:500), Foxo1 (Cell Signaling Technology, Cat# 2880 at 1:1000), p-Foxo1 ser256 (Cell Signaling Technology, Cat# 9461 at 1:1000), Foxo3 (Cell Signaling Technology, Cat# 2497 at 1:1000), p-Foxo3 ser413 (Cell Signaling Technology, Cat# 8174 at 1:1000), p-Foxo3 ser 253 (Cell Signaling Technology, Cat# 9466 at 1:1000), IRE1α (Cell Signaling Technology, Cat# 3294; at 1:1000), β1D-integrin (Millipore, MAB1900; at 1:500), LAMP1 (Development Studies Hybridoma Bank, Cat# 1D4B; at 1:100), LAMP2 (Development Studies Hybridoma Bank, Cat# ABL-93; at 1:100), LC3b (Cell Signaling Technology, Cat# 3868; at 1:1000), mTOR (Cell Signaling Technology, Cat# 2972 at 1:1000), p -mTOR ser2448 (Cell Signaling Technology, Cat# 2971 at 1:1000), p62 (Sigma, Cat# p0067 at 1:1000), LIMPII (Abcam, Cat# ab176317; at 1:1000), p-p70 S6K Thr389 (Cell Signaling Technology, Cat# 9205 at 1:1000), p70 S6K (Cell Signaling Technology, Cat# 2708 at 1:1000),PERK (Cell Signaling Technology, Cat# 3192; at 1:1000), anti-puromycin (Sigma, Cat# MABE343; at 1:1000); α-sarcoglycan (Development Studies Hybridoma Bank, Cat# IVD3(1)A9; at 1:100), β-sarcoglycan (Novus Biologicals, Cat# NBP1–90300; at 1:1000); δ-sarcoglycan (Abcam, Cat# ab137101; at 1:500); Smad2 (Cell Signaling Technology, Cat# 5339; at 1:1000); Smad3 (Abcam, Cat# ab40854; at 1:500); P-Smad2 Ser465/467/P-Smad3 Ser423/425 (Signaling Technology, Cat# 8828; at 1:1000); p-Smad3 Ser423/425 (Signaling Technology, Cat# 9520; at 1:1000); TGFβ RII (Santa Cruz Biotechnology, Cat# sc-17799; at 1:200); Thbs1 (R&D Systems, Cat# AF3074; at 1:500 and ThermoFisher Scientific, Cat# MA5–13395; at 1:1000); Thbs2 (R&D Systems, Cat# MAB1635; at 1:500); Thbs4 (R&D Systems, Cat# AF2390; at 1:500); Ubiquitin (Santa Cruz Biotechnology, Cat# sc-8017; at 1:200); and glyceraldehyde 3-phosphate dehydrogenase (Gapdh; Fitzgerald, Cat# 10R-G109A; at 1:10000) which served as overall loading control.

Techniques: Western Blot, Control, Activity Assay, Comparison, Injection, Two Tailed Test

Journal: Cell reports

Article Title: Thbs1 regulates skeletal muscle mass in a TGFβ-Smad2/3-ATF4-dependent manner

doi: 10.1016/j.celrep.2024.114149

Figure Lengend Snippet: (A) Representative western blot analysis of phospho-Smad2 (Ser465/467), total Smad2, ATF4, LC3b, p62, LAMP1, LAMP2, LIMPII, and Gapdh control in the tibialis anterior (TA) of wild-type (WT) and Thbs1 − / − mice subjected to 10 days of unilateral hindlimb denervation, compared to the sham-operated control contralateral TAs from the same mice. (B–E) Tibialis anterior (TA) weight normalized to tibia length (TL; B, D, and E) and TA myofiber cell surface area (CSA; C) after 10 days of denervation (Den.) in the indicated lines of mice, as compared to the non-denervated contralateral muscle (Ctl.) in the same mice. The legend to the left of (B) refers to all panels in the figure. Error bars denote ±SEM. The number of biologically independent animals per genotype are indicated in the graphs. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 by two-tailed unpaired Student’s t test. (F–J) Tibialis anterior (TA) weight normalized to tibia length (TL; F and I), TA myofiber cell surface area (CSA; G and J), and liver weight normalized to TL (H) from 8-week-old mice of indicated genotypes shown in the legend to the left of (B), which were fed ad libitum or fasted for 48 h. Error bars denote ±SEM. The number of biologically independent animals per genotype are indicated in the graphs. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 by one-way ANOVA and Tukey’s multiple comparison test.

Article Snippet: Primary antibodies used in this study were: Akt (Cell Signaling Technology, Cat# 9272 at 1:1000), p-Akt Ser473 (Cell Signaling Technology, Cat# 4060 at 1:1000), AMPKα (Cell Signaling Technology, Cat# 2532 at 1:1000), p-AMPKα Thr172 (Cell Signaling Technology, Cat# 2535 at 1:1000), ATF4 (Cell Signaling Technology, Cat# 11815 and LSBio LifeSpan Biosciences, Cat# LS-B6361; both at 1:1000), ATF6α (SAB SignalWayAntibody LLC, Cat# 24383; at 1:1000), BiP (Sigma, Cat# GG8918; at 1:1000), calreticulin (Cell Signaling Technology, Cat# 2891; at 1:1000), β-dystroglycan (developmental Studies Hybridoma Bank, cat# MANDAG2 clone 7D11; 1:100), p-4E-BP1 Thr37/46 (Cell Signaling Technology, Cat# 2855 at 1:1000), 4E-BP1 (Cell Signaling Technology, Cat# 9644 at 1:1000), FGF21 (R&D Systems, Cat# AF3057; at 1:500), Foxo1 (Cell Signaling Technology, Cat# 2880 at 1:1000), p-Foxo1 ser256 (Cell Signaling Technology, Cat# 9461 at 1:1000), Foxo3 (Cell Signaling Technology, Cat# 2497 at 1:1000), p-Foxo3 ser413 (Cell Signaling Technology, Cat# 8174 at 1:1000), p-Foxo3 ser 253 (Cell Signaling Technology, Cat# 9466 at 1:1000), IRE1α (Cell Signaling Technology, Cat# 3294; at 1:1000), β1D-integrin (Millipore, MAB1900; at 1:500), LAMP1 (Development Studies Hybridoma Bank, Cat# 1D4B; at 1:100), LAMP2 (Development Studies Hybridoma Bank, Cat# ABL-93; at 1:100), LC3b (Cell Signaling Technology, Cat# 3868; at 1:1000), mTOR (Cell Signaling Technology, Cat# 2972 at 1:1000), p -mTOR ser2448 (Cell Signaling Technology, Cat# 2971 at 1:1000), p62 (Sigma, Cat# p0067 at 1:1000), LIMPII (Abcam, Cat# ab176317; at 1:1000), p-p70 S6K Thr389 (Cell Signaling Technology, Cat# 9205 at 1:1000), p70 S6K (Cell Signaling Technology, Cat# 2708 at 1:1000),PERK (Cell Signaling Technology, Cat# 3192; at 1:1000), anti-puromycin (Sigma, Cat# MABE343; at 1:1000); α-sarcoglycan (Development Studies Hybridoma Bank, Cat# IVD3(1)A9; at 1:100), β-sarcoglycan (Novus Biologicals, Cat# NBP1–90300; at 1:1000); δ-sarcoglycan (Abcam, Cat# ab137101; at 1:500); Smad2 (Cell Signaling Technology, Cat# 5339; at 1:1000); Smad3 (Abcam, Cat# ab40854; at 1:500); P-Smad2 Ser465/467/P-Smad3 Ser423/425 (Signaling Technology, Cat# 8828; at 1:1000); p-Smad3 Ser423/425 (Signaling Technology, Cat# 9520; at 1:1000); TGFβ RII (Santa Cruz Biotechnology, Cat# sc-17799; at 1:200); Thbs1 (R&D Systems, Cat# AF3074; at 1:500 and ThermoFisher Scientific, Cat# MA5–13395; at 1:1000); Thbs2 (R&D Systems, Cat# MAB1635; at 1:500); Thbs4 (R&D Systems, Cat# AF2390; at 1:500); Ubiquitin (Santa Cruz Biotechnology, Cat# sc-8017; at 1:200); and glyceraldehyde 3-phosphate dehydrogenase (Gapdh; Fitzgerald, Cat# 10R-G109A; at 1:10000) which served as overall loading control.

Techniques: Western Blot, Control, Two Tailed Test, Comparison