Phospho specific antibody

SDS-PAGE and immunoblotting were performed using standard protocols as described previously [31 (link)]. Rabbit anti-human 14-3-3ζ (Santa Cruz Biotechnology, USA) was used to detect 14-3-3ζ to ensure equivalent levels of protein loading on immunoblots. The phospho-specific antibodies against various signalling proteins (Akt, ERK1/2 and PLA₂) were obtained from Cell signalling technology, USA (catologue numbers: 9271, 4370 and 2831 respectively). The secondary antibodies for immunoblotting; Cy5 goat anti-rabbit IgG and Cy3 goat anti-mouse IgG antibodies were obtained from Invitrogen, UK. Blots were visualised using a Typhoon FLA 9500 fluorimager (GE healthcare, UK). Image Quant TL software (GE Healthcare) was used for the fluorescence visualisation and analysis of protein bands.

Serum-starved cells were then stimulated with progranulin (40 nM) for 10 min. The activation of Akt and ERK1/2 was analyzed by western immunoblot using phospho-specific antibodies (Cell Signaling Technology, Beverly, MA, USA) as described [44 (link)]. Total levels of Akt and ERKs were monitored with anti-Akt and anti-ERK1/2 antibodies (Cell Signaling Technology).

Antibodies to GRP78, IRE1α, PERK, eIF2α, ATF4, Bcl-2, Bax, LC3II, LC3I, p62, and phospho-specific antibodies to p-IRE1α/p-PERK p-eIF2α and p-JNK were from Cell Signaling Technology (Beverly, MA, USA). Anticaspase 9, CHOP, BDNF, SYN, and AChR antibodies were obtained from Abcam Inc. (Cambridge, MA, USA). Anti-Beclin-1 antibody was purchased from Novus Biologicals (Littleton, San Diego, USA). Anti-FATP1 antibody was obtained from Santa Cruz Biotechnology (Dallas, Texas, CA, USA). Palmitic acid (PA) and 4-phenylbutyrate (4-PBA) were from Sigma-Aldrich Corp. (St. Louis, MO, USA). 3-Methyladenine (3-MA) was purchased from MedChemExpress (HY-19312, New Jersey, USA).

Phosphorylation of Akt/PKB (Thr 308) and Erk (Thr 202, Tyr 204) was analyzed by Western blotting with phosphospecific antibodies (Cell Signaling Technology, Leiden, The Netherlands) as described.19

Cells grown in tissue culture plates were lysed directly in plates by 200 or 100 μl of M-PER lysis buffer (Pierce) supplemented with protease and phosphatase inhibitor cocktail at 4°C for 20 min. The whole cell lysate was mixed with 2 x SDS loading buffer, boiled, and subjected to Western blotting. The PVDF membrane was blocked with 5 % NFDM (non-fat dry milk) at room temperature for around 1 h. Corresponding primary antibodies were diluted in AbDil-Tween (TBS; 2 % BSA; 0.1 % Tween-20) at 1:1000 dilution. Primary antibodies used include phospho-specific antibodies, EGFR, Akt, S6K, 4EBP1, and the HRP-linked anti-rabbit and anti-mouse IgG antibodies which were from Cell Signaling Technology. The mouse monoclonal antibodies for beta-tubulin and beta-actin were from Beijing TransGen Biotech. All primary antibodies were diluted in AbDil-Tween at 1:1000 dilution (TBS supplemented with 2% BSA and 0.1% Tween-20) and HRP conjugated secondary antibodies were diluted in TBS with 0.1 % Tween-20 and 5 % NFDM at 1:5000 dilution. Western blotting results were obtained by Bio-Rad ChemiDoc^TM XRS+ System and Beijing Tanon Fine-do X6. ImageJ software was used to quantify the protein relative level shown by Western blots.