Ab231253

Sunitinib (s7781), DC661(S8808), MG132(S2619), 3-MA(S2767), CA-074ME(S7420), Ionomycin (s7074), Puromycin(s7417) were purchased from Selleckchem (Houston, TX, USA). Fluo-3(46393), Earle’s Balanced Salt Solution (EBSS, E7510) were purchased from Sigma-Aldrich. The primary antibodies against E-Syt1(ab118805), FTH1(ab231253), TFE3 (ab196681), CTSB (ab125067), Syt7(121383), mCherry (ab183628), GAPDH (ab8245), LC3B (ab192890), GFP (ab290), HA (ab9110), mTOR (ab32028), p-mTOR (ab109268), MMP9 (ab76003), H3 (ab1791) were purchased from Abcam (Cambridge, Cambridgeshire, UK). The primary antibody of FAM14B (21573-1-AP) were purchased from Proteintech (Chicago, IL, USA). The horseradish peroxidase (HRP) labeled Goat anti-Rabbit (ab6721) and Rabbit anti-Mouse (ab6728) secondary antibodies were purchases from Abcam.

The colonic mucosa and renal tissues were collected from WT and DKO piglets. The qPCR and Western blotting analyses were performed as detailed in a previous study (19 (link)). The 18S rRNA gene was used as the internal control for target mRNA levels normalization. Primer sequences are shown in Table S2. The antibodies for Western blotting were anti-Ferritin Heavy Chain (ab231253: Abcam, Cambridge, UK), GAPDH (14C10) Rabbit MAb (2118S, Cell Signaling Technology, Danvers, MA, USA) and anti-rabbit IgG HRP-linked antibody (7074S, Cell Signaling Technology, Danvers, MA, USA).

The PAMs were lysed in RIPA lysis buffer containing a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). Total protein was harvested and analyzed for concentration via a Micro BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). The protein sample (30 µg) was loaded for electrophoresis and transferred to a nitrocellulose membrane. The membrane was immunoblotted against the primary antibody H-ferritin (ab231253; Abcam, Cambridge, UK; at 1:1000) and the fluorophore-conjugated secondary antibody (Jackson ImmunoResearch Laboratories; anti-rabbit Alexa680; at 1:3000). The band density of the target protein was normalized to the total protein detected using the ChemiDoc MP imaging system (Bio-Rad, Hercules, CA, USA). The relative expression of H-ferritin was quantified by comparing it to the average density of the CON samples using Image Lab software.