Journal: BMB Reports

Article Title: Improved efficiency of protein aggregate removal using ubiquitin with a signal peptide

doi: 10.5483/BMBRep.2025-0153

Figure Lengend Snippet: Secretion of ER-generated HA-Ub E when SP-HA-Ub is expressed in HEK293T cells. (A) Schematic representation of HA-Ub, SP-HA-Ub, and LCN2. The 20-amino-acid signal peptide (SP) originated from LCN2 was added to the N-terminus of HA-Ub. (B) Altered fate of SP-HA-Ub due to the recognition of SP by SRP. (C) SP-HA-Ub or HA-Ub construct transfected into HEK293T cells and Ub n (free Ub and Ub conjugates) and HA bands detected by immunoblotting using HEK293T cell lysates and the culture medium. For better detection of HA bands, the immunoblot with a long exposure time is also shown for SP-HA-Ub (long: 2 min; short: 7 sec). α-Tubulin (α-Tub) or Coomassie blue staining was used as a loading control.

Article Snippet: TUBA/α-Tubulin , Mouse IgG , Santa Cruz Biotechnology , SC-32293 , 1:1,000 , .

Techniques: Generated, Construct, Transfection, Western Blot, Staining, Control

Journal: BMB Reports

Article Title: Improved efficiency of protein aggregate removal using ubiquitin with a signal peptide

doi: 10.5483/BMBRep.2025-0153

Figure Lengend Snippet: Functional comparison between HA-Ub and HA-Ub E originating from SP-HA-Ub expressed in HEK293T cells. (A) Immunoblot detection of HA-Ub and HA-Ub E in HEK293T cells treated with 1 μM bortezomib (BTZ) or 100 nM bafilomycin A1 (BAF A1) for 18 h. Ub n and HA bands were assessed in cell lysates and cultured media. α-Tubulin (α-Tub) or Coomassie blue staining was used as a loading control. (B) Quantification of HA (HA-Ub and HA-Ub E conjugates) band intensities in (A) normalized to α-tubulin levels. Normalized HA band intensities were expressed as fold changes relative to non-treated controls (–; SP-HA-Ub or HA-Ub, n = 3 per group). Data are presented as the mean ± SEM from the indicated number of samples using the two-tailed unpaired t-test. # P < 0.1; *P < 0.05; **P < 0.01 vs. each non-treated control.

Article Snippet: TUBA/α-Tubulin , Mouse IgG , Santa Cruz Biotechnology , SC-32293 , 1:1,000 , .

Techniques: Functional Assay, Comparison, Western Blot, Cell Culture, Staining, Control, Two Tailed Test

Journal: BMB Reports

Article Title: Improved efficiency of protein aggregate removal using ubiquitin with a signal peptide

doi: 10.5483/BMBRep.2025-0153

Figure Lengend Snippet: Improved protein aggregate removal capability by autophagy under proteotoxic stress conditions when SP-HA-Ub is expressed in HEK293T cells. (A) Immunoblot detection of HA-Ub and HA-Ub E in HEK293T cells treated with 20 nM NaAsO 2 in the presence or absence of 2 μM torin 1 for 18 h. Ubn and HA bands were assessed in cell lysates. For better detection of HA bands, the immunoblot with a long exposure time was also shown for SP-HA-Ub (long: 5 min; short: 3 sec). α-Tubulin (α-Tub) was used as a loading control. (B) Quantification of HA (HA-Ub and HA-Ub E conjugates) band intensities in (A) normalized to α-tubulin levels. Normalized HA band intensities expressed as fold changes relative to non-treated controls (–; HA-Ub, n = 3, and SP-HA-Ub, n = 7). Data are presented as the mean ± SEM from the indicated number of samples using the two-tailed unpaired t-test. **P < 0.01 vs. each non-treated control; ns, not significant.

Article Snippet: TUBA/α-Tubulin , Mouse IgG , Santa Cruz Biotechnology , SC-32293 , 1:1,000 , .

Techniques: Western Blot, Control, Two Tailed Test

Journal: Science (New York, N.Y.)

Article Title: Mitochondria protect against an intracellular pathogen by restricting access to folate

doi: 10.1126/science.adr6326

Figure Lengend Snippet: ( A ) Immunoblot (IB) analyses of WT and eIF2α S51A AN3–12 cells that were uninfected (uninf), infected with Toxoplasma ( Toxo ), or tunicamycin (TUN)-treated (3 μg/ml) for 24 hours (h): ATF4; phospho-EIF2α (Ser51), ~38 kDa; α-Tubulin (TUBA), ~55 kDa; and TgGAP45. ( B ) Heatmap of mRNA levels of ATF4 target genes in uninf, Toxo , and TUN (3 μg/ml)-treated ES-2 cells; n=3 independent cultures. ( C and D ) WT and ATF4 KO ES-2 cells were uninf or Toxo for 24 h and analyzed by qPCR for SHMT2 and MTHFD2 . Transcripts were normalized to ACTB and are relative to WT uninf. Data are mean ± SD of n=7 independent cultures. *p<0.05; ****p<0.0001 for uninf versus Toxo by two-way ANOVA analysis. ( E ) IB of ES-2 cells as indicated: MTHFD2, ~32 kDa and TUBA. ( F, G and H ) ES-2 cells as indicated were analyzed for mtDNA by qPCR for DLOOP , mt -ND1 and mt -CTYB normalized to RUNX2 levels and relative to WT uninfected. Data are mean ± SD of n=4 independent cultures. *p<0.05; **p<0.01 for uninf versus Toxo , and #p<0.05; ##p<0.01 for WT versus MTHFD2 KO by two-way ANOVA analysis. ( I ) IB analysis of ES-2 cells as indicated at 24 hpi (MOI: 4 for Toxo ): ATF4, VCL, and TgGAP45. ( J and K) Cells treated as in ( I ) were analyzed by qPCR as in ( C and D ). Data are mean ± SD of n=4 independent cultures, *p<0.05; **p<0.01 by one-way ANOVA analysis. ( L ) IB analysis of WT and HRI KO ES-2 cells that were uninf or Toxo at 24 hpi; Toxoplasma GRA7 (TgGRA7), ~32 kDa, ( M ) IB analysis of uninf and Toxo WT and OMA1 KO mouse embryonic fibroblasts (MEFs) at 24 hpi: OMA1, ~35 kDa, ATF4, ACTB, and TgGAP45. ( N ) Toxo WT and ATF4 KO ES-2 were treated ± ISRIB (200nM) and analyzed 24 hpi via flow cytometry for Toxoplasma burden (mCherry median FI). Data are mean ± SEM of n=3 biological experiments, **p< 0.01 for DMSO versus ISRIB, #### p < 0.0001 for WT versus ATF4 KO by two-way ANOVA analysis.

Article Snippet: The following antibodies were used: ATF4 (CST #11815), VCL (CST #4650), pS6K (CST #97596), TUBA (Proteintech 66031–1-Ig), MTHFD2 (CST #41377), SHMT2 (CST #33443), MTHFD1L (Invitrogen #PA5–100158), MTHFD1 (Sigma #HPA015006), phospho-eIF2a (Ser51) (CST #9721), ACTB (CST #4970), OMA1 (Santa Cruz #515788), BIP (CST #3183), ATF6 (CST #65880), c-Myc ( CST #18583), anti-DNA (EMD Millipore #CBL186), GCN2 (CST#3302); GCN2 (abcam ab134053), TgGra45 (Dr. D Soldati; U. of Geneva), TgMAF1 and TgGRA7 (Dr. J Boothroyd; Stanford University) antisera.

Techniques: Western Blot, Infection, Flow Cytometry