Ab61213

IHC was performed to examine TGFBR2 and hTERT expression in cervical tissues by using primary antibodies against TGFBR2 (1:100 dilution; ab61213, Abcam, USA) and hTERT (1:25 dilution; ab183105, Abcam, USA), according to the reported procedures45 (link). A semi-quantitative estimate was composed based on the staining intensity and the extent of stained cells; the evaluation criteria were as follows: intensity [categorized as 0 (no staining), 1 (weak staining, light yellow), 2 (moderate staining, yellowish brown), or 3 (strong staining, brown)] and the percentage of positively stained cells [scored as 0 (0–5% positive), 1 (6–25%), 2 (26–50%), 3 (51–75%), or 4 (>75%)]. Finally, intensity was multiplied by the percentage of positively stained cells. The results were assessed independently by two investigators who were blinded to the patient’s clinicopathological outcomes. When the investigators originally proposed different results, they reached a consensus. The average result of the two independent investigators was the final reported staining score. Both scores of all the samples were clarified into four categories: 0–1 represented negative expression (−), 2–4 weak expression (+), 5–8 moderate expression (++), and ≥9 high expression (+++).

IHC assay was performed and quantitatively analyzed as previously described [45 (link), 46 (link)]. Paraffin-embedded NSCLC serial sections were analyzed with antibodies against TGFBR2 (Abcam, ab61213), USP15 (Abcam, ab71713) and SMAD3 (Abcam, ab28379), respectively. The degree of immunostaining of target proteins was evaluated and scored based on the proportions of tumor cells stained positively and the intensity of the staining by two independent observers.

Cells were fixed and permeabilized with Perm/Wash Buffer (Cat. # 554723, BD Biosciences). Samples were blocked with Image-iT FX signal enhancer for 30 min and incubated for 2 hours at room temperature with primary antibodies combined with reagents of Zenon Alexa Fluor 488 (green) or 555 (red) labeling kit (Invitrogen). To detect Ki-67 in PCa cells co-cultured with MC3T3-E1 cells in vitro, human Ki-67 (cat. # ab15580, Abcam) and HLA-ABC (Cat. # 311402, BioLegend) antibodies were used as primary antibodies. To detect Ki-67 in PCa cells in bone marrow sections, human Ki-67 (cat. # ab15580, Abcam) and pan cytokeratin (cat. # ab867364, Abcam) antibodies were used as the primary antibody. To detect human TGFBR2 and TGFBR3, human TGFBR2 (cat. # ab61213, Abcam) and human TGFBR3 (cat. # ab78421, Abcam) antibodies were used as the primary antibody. After washing with PBS, the slides were mounted with ProLong Gold antifade reagent with DAPI (Invitrogen). Images were taken with Nikon A-1-B confocal microscope.

Geniposide (≥98% purity, as detected by high-performance liquid chromatography analysis) was purchased from Shanghai Winherb Medical Co. (Shanghai, China). TGF-β (T7039) was obtained from Sigma-Aldrich (St. Louis, MO, United States). ISO was purchased from Sigma-Aldrich Co. EX-527 was purchased from MedchemExpress (Sollentuna, Sweden). Antibodies against the following proteins were purchased from Abcam: TGF-β1 (ab66043), SIRT1 (ab110304), gp91 (ab80508), 4-hydroxynonenal (4HNE) (ab46545), thioredoxin2 (ab26320), α-SMA (ab5694), GRP78 (ab955), XBP-1 (ab37152), TGF beta Receptor I (ab31013), TGF beta Receptor II (ab61213), and acetyl-lysine (ab80178). Antibodies against the following proteins were purchased from Cell Signaling Technology: phosphorylated (P-)Smad3 (8769), total (T-)Smad3 (9513s), and GAPDH (2118). The antibodies against P-PERK (sc-32577) and T-PERK (sc13073) were purchased from Santa Cruz Biotechnology. The antibody against ATF6 (15794-1-AP) was purchased from Proteintech Group. The secondary antibodies used in this study were acquired from LI-COR Biosciences (used at 1:10,000 dilution). The GT VisionTM+Detection System/Mo&Rb reagent for immunohistochemistry was obtained from Gene Technology (Shanghai, China). The Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody for immunofluorescence was purchased from LI-COR Biosciences.

Histopathological analysis was performed on formalin‐fixed lung biopsy tissues from patients with IPF and mice by hematoxylin and eosin (H&E) and Masson's trichrome staining. Primary antibodies were TGF‐β1 (ab92486) and TGF‐βR2 (ab61213; Abcam, USA). The pathological scores of alveolitis at 7 days and fibrosis at 21 days in BLM‐induced mice treatment were calculated based on the method as described previously.¹⁹ (link) The alveolitis and fibrosis scores were performed blindly by two senior pathologists independently.