Anti phospho ampk thr172

The following plasmids were used for transfection: pcDNA3.1(-)-GFP-LC3 [47 (link)], mRFP-GFP-LC3 construct [48 (link)]. hPC1-expressing plasmid WT (REP10) was a gift from Gregory Germino (Addgene plasmid #21368) [49 (link)] as was hPC2-expressing plasmid (M-PKD2 (OF2-3); Addgene plasmid #21370) [21 (link)].
The following siRNAs were used: siRNA against mouse Pkd2 (AM16708, assay ID 150154, Thermo Fischer Scientific), siRNA against mouse BECN1 (4390771, Ambion, Woolston, UK) and negative control DsiRNA (51-01-14-04, IDT).
The following antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA): anti-β-Actin (4970), anti-FoxO1 (14952), anti-ULK1 (8054), anti-phospho-ULK1 (Ser555) (5869), anti-phospho-ULK1 (Ser757) (14202), anti-phospho-ULK1 (Ser317) (3776), anti-AMPK (5832), anti-phospho AMPK (Thr172) (50081), anti-S6 (2217) and anti-p-S6 (4858). Anti-PC1 (sc-130554), anti-PC2 (sc-28331) and anti-SV40 LT were from Santa Cruz Biotechnology (Dallas, TX, USA), anti-Sqstm1 (P0067) and anti-EpCam (HPA026761) from Sigma-Aldrich (Saint-Louis, MO, USA, anti-LC3 from Nanotools (clone 5F10; München, Germany), anti-PgP from Abcam (ab170904; Cambridge, UK) and anti-AQP1 from Novus Biologicals (84488; Abingdon, UK). Secondary anti-rabbit and anti-mouse antibodies were from Bio-Rad Laboratories (Hercules, CA, USA).

Protein samples were extracted from liver tissues and cultured cells as previously reported [28 (link)]. Equal amounts of protein (30–40 μg) were separated by 8%–15% SDS-PAGE and then transferred onto PVDF membranes. For immunoblot analysis of IL-1β levels in the cell culture medium, supernatants were collected for protein extraction as previously reported [35 (link)]. The following primary antibodies were used: anti-IL-1β (number 12242), anti-AMPK (number 2532), anti-phospho-AMPK Thr¹⁷² (number 2535), anti-phospho-GSK3β Ser⁹ (number 5558), anti-acetyl coenzyme A carboxylase (ACC, number 3676), anti-ACC Ser⁷⁹ (number 3661) (Cell Signaling Technology, Beverly, MA, USA), anti-phospho-Akt Ser⁴⁷³ (number 2118-1) (Epitomics, Burlingame, CA, USA), anti-caspase-1 (number 22915-1-AP), anti-NLRP3 (number 19771-1-AP), anti-GSK3β (number 22104-1-AP) (Proteintech Group, Chicago, IL, USA), anti-TXNIP (number sc-67134) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-Akt (number A0001)(ABclonal Biotech Co. Ltd., Cambridge, MA, USA). Goat anti-rabbit (number A21020) and goat anti-mouse (number A21010) secondary antibodies were purchased from Abbkine (Redlands, CA, USA).

For the western blot analysis, 50 mg of LV myocardial tissue was homogenized on ice in radioimmunoprecipitation lysis buffer containing a complete protease inhibitor cocktail (Roche Diagnostics). The homogenates were centrifuged to collect the supernatants. The protein concentration was determined using a BCA protein assay kit (Thermo Scientific). LV myocardial samples were subjected to SDS-PAGE and transferred to polyvinylidene difluoride membranes. Membranes were blocked with 5% BSA in PBS plus Tween 20 at room temperature for 2 h, and then incubated with anti-PI3K (P110α), anti-Akt (Ser172), anti-phospho-Akt (Ser172), anti-GLUT4, anti-AMPK (Thr172), anti-phospho-AMPK (Thr172), and anti-eNOS antibodies (Cell Signaling Technology, Danvers, MA) at 4 °C overnight. Membranes were subsequently washed and incubated with the relevant secondary horseradish peroxidase-conjugated antibodies (1:5000; ZSGB-BIO, Beijing, China) for 90 min. β-actin (mouse anti-β-actin; 1:500; ZSGB-BIO) was used as the loading control. Intensity was measured using Quantity One densitometric software (Bio-Rad Laboratories, Hercules, CA), as described previously [18 (link)].

Anti-AMP-activated protein kinase (AMPK; #5831; 1:1000), anti-phospho-AMPK [Thr172] (#2535; 1:1000), anti-ACC (#3676; 1:1000), anti-phospho-ACC [Ser79] (#11818; 1:1000), anti-S6K (#2708; 1:1000), and anti-phopho-S6K [Thr389] (#9234; 1:1000) were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-SCD1 (ab39969; 1:500), and anti-actin (ab6276; 1:5000) were purchased from Abcam (Cambridge, MA, USA). Anti-microtubule-associated protein 1A/1B-light chain 3 (LC3; PM036; 1:2000) was purchased from MBL (Nagoya, Japan). Secondary anti-rabbit (#7074; 1:2000) and anti-mouse (715-035-151; 1:10000) antibodies were purchased from Cell Signaling Technology and Jackson ImmunoResearch (West Grove, PA, USA), respectively.