Journal: The EMBO Journal

Article Title: Micropeptide hSPAR regulates glutamine levels and suppresses mammary tumor growth via a TRIM21-P27KIP1-mTOR axis

doi: 10.1038/s44318-024-00359-z

Figure Lengend Snippet: Reagents and tools table

Article Snippet: The membranes were blocked in 5% BSA (Sangon, China) for 1 h at room temperature, and then incubated at 4 °C overnight with primary antibody GAPDH (Proteintech, USA, 60004-1-Ig), hSPAR (HuaBio, China), Flag (Abcam, UK, ab205606), β-Tubulin (Proteintech, USA, 10068-1-AP), FIBRILLARIN (Proteintech, USA, 16021-1-AP), ATPV1A (Proteintech, USA, 14418-1-AP), LAMP2 (CST, USA, 49067), TRIM21 (Proteintech, USA, 67136-1-Ig), P27KIP1 (Proteintech, USA, 25614-1-AP), phospho-P27KIP1 (Abcam, USA, ab75908), SKP2 (Proteintech, USA, 15010-1-AP), SLC7A1 (Proteintech, USA, 14195-1-AP), SLC7A5 (Proteintech, USA, 28670-1-AP), phospho-mTOR (CST, USA, 5536), mTOR (CST, USA, 2983), phospho-S6K (CST, USA, 9234), S6K (CST, USA, 2708), phospho-S6 (CST, USA, 2211), S6 (CST, USA, 2217), phospho-AKT (CST, USA, 4060), AKT (CST, USA, 9272), Ubiquitin (Abcam, UK, ab134953), SLC38A2 (ImmunoWay, USA, YT4354), LAMTOR1(CST, USA, 8975), LAMTOR2 (CST, USA, 8145), LAMTOR3 (Proteintech, USA, 14492-1-AP), LAMTOR4 (CST, USA, 13140), LAMTOR5 (Proteintech, USA,11937-1-AP), RagA (CST, USA, 4357S) and TAT (Abcam, UK, ab42359).

Techniques: Recombinant, Sequencing, Modification, Membrane, Lysis, Transfection, Magnetic Beads, Software, Cytometry, In Vitro, cDNA Synthesis, Plasmid Preparation, Isolation, Mutagenesis, Silver Staining

Journal: Biology of reproduction

Article Title: Transforming growth factor beta3 regulates the dynamics of Sertoli cell tight junctions via the p38 mitogen-activated protein kinase pathway.

doi: 10.1095/biolreprod.102.011387

Figure Lengend Snippet: FIG. 1. A schematic drawing illustrates the four signaling pathways utilized by TGFb to affect cellular function. This diagram was prepared based on reviews [18, 19, 21]. Original articles that describe the discovery of the key molecules depicted in these pathways can be found in these reviews. MEKKs, MAP/ERK kinase kinases, which include MEKK1, MEKK2, MEKK3, and others; JNK, c-Jun NH2-terminal kinase also known as Jun kinase or stress-activated protein kinase, SAPK; JNKK, c-Jun NH2-terminal kinase kinase; MKK3, MAP kinase kinase 3; MEK1/2, MAP/ERK kinase 1 and MAP/ERK kinase 2; ERK1/2, extracellular signal-regulated kinase 1 and extracellular signal-regulated kinase 2.

Article Snippet: Antibodies against TGFb3 (cat. no. sc-82, lot. no. H280), MEKK2 (cat. no. sc-1088, lot. no. A181), Rac2 (cat. no. sc-96, lot. no. G231), and N-Ras (cat. no. sc-31, lot. no. D111) were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Protein-Protein interactions, Cell Function Assay

Journal: Biology of reproduction

Article Title: Transforming growth factor beta3 regulates the dynamics of Sertoli cell tight junctions via the p38 mitogen-activated protein kinase pathway.

doi: 10.1095/biolreprod.102.011387

Figure Lengend Snippet: FIG. 4. Relative expression of the TGFb- upstream signal transducers in Sertoli and germ cells isolated from 20-day-old rat testes. A) Semiquantitative RT-PCR was performed to assess the steady-state mRNA levels of Smad2, Cdc42, Rac2, MEKK2, and N-Ras in Sertoli and germ cells. Im- munoblots were performed in parallel ex- periments to assess the relative protein levels of these signal transducers in Sertoli and germ cells using the corresponding specific antibodies (see Materials and Methods). (C). B) This figure shows the corresponding densitometrically scanned results using autoradiograms or immuno- blots such as those shown in A and C. Re- sults are expressed as mean 6 SD using three batches of cells from three different experiments normalized against S16. Each experiment had triplicate cultures. These analyses revealed that results of immuno- blots are consistent with the RT-PCR data. Statistical analysis was performed by Stu- dent t-test comparing germ cells with the corresponding Sertoli cells, which was ar- bitrarily set at one. *, Significantly different from Sertoli cells by Student t-test, P , 0.05; **, significantly different from Sertoli cells by Student t-test, P , 0.01.

Article Snippet: Antibodies against TGFb3 (cat. no. sc-82, lot. no. H280), MEKK2 (cat. no. sc-1088, lot. no. A181), Rac2 (cat. no. sc-96, lot. no. G231), and N-Ras (cat. no. sc-31, lot. no. D111) were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Expressing, Isolation, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Biology of reproduction

Article Title: Transforming growth factor beta3 regulates the dynamics of Sertoli cell tight junctions via the p38 mitogen-activated protein kinase pathway.

doi: 10.1095/biolreprod.102.011387

Figure Lengend Snippet: FIG. 5. Developmental regulation of the steady-state mRNA levels of the TGFb up- stream signal transducers in Sertoli (A, B) and germ (C, D) cells. RT-PCR was per- formed to assess the steady-state mRNA levels of Smad2, Cdc42, Rac2, MEKK2, and N-Ras in Sertoli cells (A) and germ cells (C) during maturation. B, D) The cor- responding densitometrically scanned re- sults using autoradiograms such as those shown in A and C. Results are expressed as mean 6 SD using two batches of cells normalized against S16 from two different experiments. Each experiment had tripli- cate cultures. ns, Not significantly different from cultures isolated from rats at 20 days of age in B and 5 or 10 days of age in D, which was arbitrarily set at one, by Stu- dent t-test; *, significantly different by Stu- dent t-test, P , 0.05; **, significantly dif- ferent by Student t-test, P , 0.01; nd, not detectable.

Article Snippet: Antibodies against TGFb3 (cat. no. sc-82, lot. no. H280), MEKK2 (cat. no. sc-1088, lot. no. A181), Rac2 (cat. no. sc-96, lot. no. G231), and N-Ras (cat. no. sc-31, lot. no. D111) were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Isolation

Journal: Biology of reproduction

Article Title: Transforming growth factor beta3 regulates the dynamics of Sertoli cell tight junctions via the p38 mitogen-activated protein kinase pathway.

doi: 10.1095/biolreprod.102.011387

Figure Lengend Snippet: FIG. 6. Changes in the steady-state mRNA levels of TGFb upstream signal transducers in the testis during develop- ment. A) Semiquantitative RT-PCR was performed to assess the steady-state mRNA levels of Smad2, Cdc42, Rac2, MEKK2, and N-Ras in testes during maturation. B) This panel shows the corresponding densi- tometrically scanned results using autora- diograms such as those shown in A. Re- sults are expressed as mean 6 SD using testes from three different rats normalized against S16. ns, Not significantly different from rats at 5 days of age, which was arbi- trarily set at one, by Student t-test; *, signif- icantly different by Student t-test, P , 0.05; **, significantly different by Student t-test, P , 0.01.

Article Snippet: Antibodies against TGFb3 (cat. no. sc-82, lot. no. H280), MEKK2 (cat. no. sc-1088, lot. no. A181), Rac2 (cat. no. sc-96, lot. no. G231), and N-Ras (cat. no. sc-31, lot. no. D111) were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Reverse Transcription Polymerase Chain Reaction

Journal: Biology of reproduction

Article Title: Transforming growth factor beta3 regulates the dynamics of Sertoli cell tight junctions via the p38 mitogen-activated protein kinase pathway.

doi: 10.1095/biolreprod.102.011387

Figure Lengend Snippet: FIG. 8. Change in the steady-state mRNA and protein levels of the TGFb upstream signal transducers when the Sertoli cell TJ barrier was assembled in vitro in the ab- sence (control) and presence (test) of TGFb3. Sertoli cells (0.5 3 106 cells/cm2) cultured on Matrigel-coated dishes in the absence (A, C, E) or presence (B, D, F) of TGFb3 (3 ng/ml) were terminated by RNA STAT-60 or lysed in SDS sample buffer at specified time points. Cell lysates (;200 mg protein) from each time point were re- solved by SDS-PAGE under reducing con- ditions using 10% T SDS-polyacrylamide gels. Immunoblotting was performed to as- sess changes in the levels of Smad2, Cdc42, Rac2, N-Ras, and MEKK2 in the absence (A, E) and presence (B, F) of re- combinant TGFb3 (3 ng/ml). RT-PCR was performed to assess changes in the MEKK2 steady-state mRNA level in the absence (C) or presence (D) of recombinant TGFb3 (3 ng/ml). G, H) Corresponding densito- metrically scanned results using autoradio- grams and immunoblots shown in C–F. Results are expressed as mean 6 SD from three separate experiments using different batches of cells and normalized against S16. Each time point had duplicate cul- tures. ns, Not significantly different by AN- OVA, in which each sample at a given time point was compared with samples of all other time points within the same ex- perimental group; *, significantly different by ANOVA, P , 0.01; D, days.

Article Snippet: Antibodies against TGFb3 (cat. no. sc-82, lot. no. H280), MEKK2 (cat. no. sc-1088, lot. no. A181), Rac2 (cat. no. sc-96, lot. no. G231), and N-Ras (cat. no. sc-31, lot. no. D111) were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: In Vitro, Control, Cell Culture, SDS Page, Western Blot, Reverse Transcription Polymerase Chain Reaction, Recombinant